Anti-folate receptor alpha antibodies and uses thereof

ABSTRACT

Described herein are antibodies, and antigen-binding fragments thereof, that are specific for folate receptor alpha, related polynucleotides, expression vectors, and cells that express the described antibodies. Also provided are methods of using the described antibodies, and antigen-binding fragments thereof, and related kits. Provided herein are also methods for diagnosing cancers, such as breast cancer, thyroid cancer, colorectal cancer, endometrial cancer, fallopian tube cancer, ovarian cancer, or lung cancer, using the described antibodies, and antigen-binding fragments thereof. The methods involve determining the amount of folate receptor alpha in a sample derived from a subject and comparing this level with the level of folate receptor alpha in a control sample or reference sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/800,575, filed Mar. 13, 2013, now U.S. Pat. No. 8,834,877, which is adivisional of U.S. application Ser. No. 13/548,775, filed Jul. 13, 2012,now U.S. Pat. No. 8,475,795, which claims the benefit of U.S.provisional application No. 61/508,444, filed Jul. 15, 2011; U.S.provisional application No. 61/604,412, filed Feb. 28, 2012; and U.S.provisional application No. 61/604,954, filed Feb. 29, 2012. Each ofthese applications is incorporated by reference herein in its entirety.

TECHNICAL FIELD

The subject matter provided herein relates to folate receptor alpha(FRα)-specific antibodies as well as methods of producing and using theantibodies.

BACKGROUND

In humans, the high affinity receptor for folate comes in four isoforms:alpha, beta, gamma, and delta. The alpha, beta and delta forms aretypically bound to the membranes of cells by a glycosylphosphatidylinositol (GPI) anchor. They recycle between extracellularand endocytic compartments and are capable of transporting folate intothe cell. Soluble forms of folate receptor may be derived by the actionof proteases or phospholipase on membrane anchored folate receptors.

Folate receptor alpha (also referred to as FRα, FR-alpha, FOLR-1 orFOLR1) is expressed in a variety of epithelial tissues, including thoseof the choroid plexus, lung, thyroid, kidney, uterus, breast, Fallopiantube, epididymis, and salivary glands. Weitman, S D et al., Cancer Res52: 3396-3401 (1992); Weitman S D et al., Cancer Res 52: 6708-6711(1992). Overexpression of FRα has been observed in various cancers,including lung cancer (e.g., carcinoid tumors, and non-small cell lungcancers, such as adenocarcinomas); mesothelioma; ovarian cancer; renalcancer; brain cancer (e.g., anaplastic ependymoma, cerebellar juvenilepilocytic astrocytoma, and brain metastases); cervical cancer;nasopharyngeal cancer; mesodermally derived tumor; squamous cellcarcinoma of the head and neck; endometrial cancer; papillary serous andendometrioid adenocarcinomas of the ovary, serous cystadenocarcinomas ofthe ovary, breast cancer; bladder cancer; pancreatic cancer; bone cancer(e.g., high-grade osteosarcoma); pituitary cancer (e.g., pituitaryadenomas); colorectal cancer and medullary thyroid cancer. See e.g.,U.S. Pat. No. 7,754,698; U.S. Patent Application No. 2005/0232919; Intl.Publ. No. WO 2009/132081; Bueno R et al., J of Thoracic andCardiovascular Surgery, 121(2): 225-233 (2001); Elkanat H & Ratnam M.Frontiers in Bioscience, 11, 506-519 (2006); Basal et al., PLoS ONE,4(7):6292 (2009); Fisher R E J Nucl Med, 49: 899-906 (2008); Franklin, WA et al., Int J Cancer, Suppl 8: 89-95 (1994); Hartmann L C et al., IntJ Cancer 121: 938-942 (2007); Iwakiri S et al., Annals of SurgicalOncology, 15(3): 889-899 (2008); European patent publication EP 2199796,Parker N. et al., Analytical Biochemistry, 338: 284-293 (2005); Weitman,S D et al., Cancer Res 52: 3396-3401 (1992); Saba N F et al., Head Neck,31(4): 475-481 (2009); Yang R et al., Clin Cancer Res 13: 2557-2567(2007). In some types of cancers (e.g., squamous cell carcinoma of thehead and neck), a high level of FRα expression is associated with a poorprognosis, whereas in other types of cancers (e.g., non-small-cell lungcancers), a higher level of FRα expression is associated with a morefavorable prognosis. See, e.g., Iwakiri S et al., Annals of SurgicalOncology, 15(3): 889-899; Saba N F et al., Head Neck, 31(4): 475-481(2009).

Earlier detection of cancer improves survival rates and quality of life.To improve the likelihood of early detection and treatment, a pressingneed exists for non-invasive methods for diagnosing FRα-expressingcancers and for monitoring existing FRα-expressing cancers.

SUMMARY

Provided herein are antibodies that specifically bind to FRα. Alsodescribed are related polynucleotides capable of encoding the providedantibodies, cells expressing the provided antibodies, as well asassociated vectors and detectable antibody labels. In addition, methodsof using the provided antibodies are described. For example, theprovided antibodies may be used to diagnose cancer; monitor cancerprogression, regression, or stable disease; develop a prognosis forcancer in a subject; to determine whether or not a patient should betreated for cancer, or to determine whether or not a subject isafflicted with FRα-expressing cancer and thus may be amenable totreatment with a FRα-specific anti-cancer therapeutic.

Folate Receptor Alpha (FRα)-Specific Antibodies

Described herein are isolated antibodies and antigen-binding fragmentsthat specifically bind to FRα. In some embodiments, the antibodies orantigen-binding fragments are murine IgG, or derivatives thereof.

In some embodiments, antibodies or antigen-binding fragments may includea light chain CDR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 2. In some embodiments, antibodies orantigen-binding fragments may include a light chain CDR2 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 3. Insome embodiments, antibodies or antigen-binding fragments may include alight chain CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 4. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 6. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 7. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 8. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 2; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 3; and a CDR3 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 4. The antibodies orantigen-binding fragments may include a heavy chain having a CDR1 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 6;a CDR2 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 7; and a CDR3 amino acid sequence substantially the same as,or identical to, SEQ ID NO: 8. The antibodies or antigen-bindingfragments may include a light chain having a CDR1 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 2; a CDR2 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 3;and a CDR3 amino acid sequence substantially the same as, or identicalto, SEQ ID NO: 4, and also have a heavy chain having a CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 6; aCDR2 amino acid sequence substantially the same as, or identical to, SEQID NO: 7; and a CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 8.

The described antibodies or antigen-binding fragments may include alight chain variable domain that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 5. In someembodiments, an isolated polynucleotide that includes a sequencesubstantially the same as, or identical to, SEQ ID NO: 37 may encodethis light chain variable domain amino acid sequence. The describedantibodies or antigen-binding fragments may include a heavy chainvariable domain that includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 9. In some embodiments, an isolatedpolynucleotide that includes a sequence substantially the same as, oridentical to, SEQ ID NO: 41 may encode this heavy chain variable domainamino acid sequence. The described antibodies or antigen-bindingfragments may include a light and a heavy chain variable domains,wherein the light chain variable domain includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 5, and the heavychain variable domain includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 9. In some embodiments are providedthe 9F3.H9.H3.H3.B5.G2 (9F3) antibody or antigen-binding fragmentsthereof, capable of binding either a native or nonreduced forms of FRα.

In some embodiments, the 9F3 antibody is produced by antibody-producingcells deposited with the American Type Culture Collection (10801University Blvd., Manassas, Va. 20110-2209) on May 19, 2011 and havebeen assigned Accession No. PTA-11887. In some embodiments, theantibodies, or antigen-binding fragments thereof, have the bindingaffinity for FRα of the antibodies produced by the depositedantibody-producing cells. In some embodiments, the disclosed antibodies,or antigen-binding fragments thereof, comprise the light and heavy chainCDRs of the antibodies produced by the deposited antibody-producingcells. In some embodiments, the antibodies, or antigen-binding fragmentsthereof, comprise the light and heavy chain variable regions of theantibodies produced by the deposited antibody-producing cells.

Also disclosed are isolated polynucleotides that encode antibodies orantigen-binding fragments that specifically bind to native or nonreducedforms of FRα. In some embodiments, the isolated polynucleotides encodean antibody or antigen-binding fragment thereof having a light chainCDR1 sequence substantially the same as, or identical to, SEQ ID NO: 2,for example SEQ ID NO: 34. In some embodiments, the isolatedpolynucleotides encode an antibody or antigen-binding fragment thereofhaving a light chain CDR2 substantially the same as, or identical to,SEQ ID NO: 3, for example SEQ ID NO: 35. In some embodiments, theisolated polynucleotides encode an antibody or antigen-binding fragmentthereof having a light chain CDR3 substantially the same as, oridentical to, SEQ ID NO: 4, for example SEQ ID NO: 36. In someembodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR1 substantiallythe same as, or identical to, SEQ ID NO: 6, for example SEQ ID NO: 38.In some embodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR2 substantiallythe same as, or identical to, SEQ ID NO: 7, for example SEQ ID NO: 39.In some embodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR3 substantiallythe same as, or identical to, SEQ ID NO: 8, for example SEQ ID NO: 40.The isolated polynucleotides may encode an antibody or antigen-bindingfragment thereof having a light chain with a CDR1 substantially the sameas, or identical to, SEQ ID NO: 2, for example SEQ ID NO: 34; a CDR2substantially the same as, or identical to, SEQ ID NO: 3, for exampleSEQ ID NO: 35; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 4, for example SEQ ID NO: 36. The isolated polynucleotidesmay encode an antibody or antigen-binding fragment thereof having aheavy chain CDR1 substantially the same as, or identical to, SEQ ID NO:6, for example SEQ ID NO: 38; a CDR2 substantially the same as, oridentical to, SEQ ID NO: 7, for example SEQ ID NO: 39; and a CDR3substantially the same as, or identical to, SEQ ID NO: 8, for exampleSEQ ID NO: 40. The isolated polynucleotides may encode an antibody orantigen-binding fragment thereof having a light chain CDR1 substantiallythe same as, or identical to, SEQ ID NO: 2, for example SEQ ID NO: 34; aCDR2 encoded by a nucleotide sequence substantially the same as, oridentical to, SEQ ID NO: 3, for example SEQ ID NO: 35; and a CDR3encoded by a nucleotide sequence substantially the same as, or identicalto, SEQ ID NO: 4, for example SEQ ID NO: 36; and a heavy chain CDR1substantially the same as, or identical to, SEQ ID NO: 6, for exampleSEQ ID NO: 38; a CDR2 substantially the same as, or identical to, SEQ IDNO: 7, for example SEQ ID NO: 39; and a CDR3 substantially the same as,or identical to, SEQ ID NO: 8, for example SEQ ID NO: 40.Antigen-binding arrangements of CDRs may also be engineered usingantibody-like proteins as CDR scaffolding. Such engineeredantigen-binding proteins are within the scope of the disclosure.

Polynucleotides described herein may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 5, for example SEQ ID NO: 37. In someembodiments the described isolated polynucleotides may encode antibodiesor antigen-binding fragments that have a heavy chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 9, for example SEQ ID NO: 41. In someembodiments the described isolated polynucleotides may encode antibodiesor antigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 5, for example SEQ ID NO: 37; and a heavychain variable domain segment that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 9, for exampleSEQ ID NO: 41. The isolated polynucleotides capable of encoding thevariable domain segments provided herein may be included on the same, ordifferent, vectors to produce an antibodies or antigen-bindingfragments. Polynucleotides described herein may encode the 9F3 antibodyor antigen-binding fragments thereof, capable of binding native ornonreduced forms of FRα.

Described herein are isolated antibodies and antigen-binding fragmentsthat specifically bind to native or nonreduced forms of FRα. In someembodiments, the antibodies or antigen-binding fragments are murine IgG,or derivatives thereof. While the antibodies or antigen-bindingfragments may be human, humanized, or chimeric, the antibodies orantigen-binding fragments exemplified herein are murine. In someembodiments, antibodies or antigen-binding fragments may include a lightchain CDR1 amino acid sequence substantially the same as, or identicalto, SEQ ID NO: 10. In some embodiments, antibodies or antigen-bindingfragments may include a light chain CDR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 11. In someembodiments, antibodies or antigen-binding fragments may include a lightchain CDR3 amino acid sequence substantially the same as, or identicalto, SEQ ID NO: 12. In some embodiments, antibodies or antigen-bindingfragments may include a heavy chain CDR1 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 14. In someembodiments, antibodies or antigen-binding fragments may include a heavychain CDR2 amino acid sequence substantially the same as, or identicalto, SEQ ID NO: 15. In some embodiments, antibodies or antigen-bindingfragments may include a heavy chain CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 16. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 10; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 11; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 12. Theantibodies or antigen-binding fragments may include a heavy chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 14; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 15; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 16. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 10; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 11; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 12, and also havea heavy chain having a CDR1 amino acid sequence substantially the sameas, or identical to, SEQ ID NO: 14; a CDR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 15; and a CDR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 16.

The described antibodies or antigen-binding fragments may include alight chain variable domain that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 13. In someembodiments, an isolated polynucleotide that includes a sequencesubstantially the same as, or identical to, SEQ ID NO: 45 may encodethis light chain variable domain amino acid sequence. The describedantibodies or antigen-binding fragments may include a heavy chainvariable domain that includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 17. In some embodiments, anisolated polynucleotide that includes a sequence substantially the sameas, or identical to, SEQ ID NO: 49 may encode this heavy chain variabledomain amino acid sequence. The described antibodies or antigen-bindingfragments may include a light and a heavy chain variable domains,wherein the light chain variable domain includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 13, and the heavychain variable domain includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 17. In some embodiments areprovided the 19D4.B7 (19D4) antibody or antigen-binding fragmentsthereof, capable of binding either a native or nonreduced forms of FRα.

In some embodiments, the 19D4 antibody is produced by antibody-producingcells deposited with the American Type Culture Collection (10801University Blvd., Manassas, Va. 20110-2209) on May 19, 2011 and havebeen assigned Accession No. PTA-11884. In some embodiments, theantibodies, or antigen-binding fragments thereof, have the bindingaffinity for FRα of the antibodies produced by the depositedantibody-producing cells. In some embodiments, the disclosed antibodies,or antigen-binding fragments thereof, comprise the light and heavy chainCDRs of the antibodies produced by the deposited antibody-producingcells. In some embodiments, the antibodies, or antigen-binding fragmentsthereof, comprise the light and heavy chain variable regions of theantibodies produced by the deposited antibody-producing cells.

Also disclosed are isolated polynucleotides that encode antibodies orantigen-binding fragments that specifically bind to either a native ornonreduced forms of FRα. In some embodiments, the isolatedpolynucleotides encode an antibody or antigen-binding fragment thereofhaving a light chain CDR1 sequence substantially the same as, oridentical to, SEQ ID NO: 10, for example SEQ ID NO: 42. In someembodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR2 substantiallythe same as, or identical to, SEQ ID NO: 11, for example SEQ ID NO: 43.In some embodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR3 substantiallythe same as, or identical to, SEQ ID NO: 12, for example SEQ ID NO: 44.In some embodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR1 substantiallythe same as, or identical to, SEQ ID NO: 14, for example SEQ ID NO: 46.In some embodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR2 substantiallythe same as, or identical to, SEQ ID NO: 15, for example SEQ ID NO: 47.In some embodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR3 substantiallythe same as, or identical to, SEQ ID NO: 16, for example SEQ ID NO: 48.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a light chain with a CDR1 substantially the same as, oridentical to, SEQ ID NO: 10, for example SEQ ID NO: 42; a CDR2substantially the same as, or identical to, SEQ ID NO: 11, for exampleSEQ ID NO: 43; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 12, for example SEQ ID NO: 44. The polynucleotides may encodean antibody or antigen-binding fragment thereof having a heavy chainCDR1 substantially the same as, or identical to, SEQ ID NO: 14, forexample SEQ ID NO: 46; a CDR2 substantially the same as, or identicalto, SEQ ID NO: 15, for example SEQ ID NO: 47; and a CDR3 substantiallythe same as, or identical to, SEQ ID NO: 16, for example SEQ ID NO: 48.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a light chain CDR1 substantially the same as, oridentical to, SEQ ID NO: 10, for example SEQ ID NO: 42; a CDR2 encodedby a nucleotide sequence substantially the same as, or identical to, SEQID NO: 11, for example SEQ ID NO: 43; and a CDR3 encoded by a nucleotidesequence substantially the same as, or identical to, SEQ ID NO: 12, forexample SEQ ID NO: 44; and a heavy chain CDR1 substantially the same as,or identical to, SEQ ID NO: 14, for example SEQ ID NO: 46; a CDR2substantially the same as, or identical to, SEQ ID NO: 15, for exampleSEQ ID NO: 47; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 16, for example SEQ ID NO: 48. Antigen-binding arrangementsof CDRs may also be engineered using antibody-like proteins as CDRscaffolding. Such engineered antigen-binding proteins are within thescope of the disclosure.

Polynucleotides described herein may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 13, for example SEQ ID NO: 45. In someembodiments the described polynucleotides may encode antibodies orantigen-binding fragments that have a heavy chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 17, for example SEQ ID NO: 49. In someembodiments the described polynucleotides may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 13, for example SEQ ID NO: 45; and a heavychain variable domain segment that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 17, for exampleSEQ ID NO: 49. The polynucleotides capable of encoding the variabledomain segments provided herein may be included on the same, ordifferent, vectors to produce an antibodies or antigen-bindingfragments. Polynucleotides described herein may encode the 19D4 antibodyor antigen-binding fragments thereof, capable of binding native ornonreduced forms of FRα.

Described herein are isolated antibodies and antigen-binding fragmentsthat specifically bind to FRα, capable of binding native or nonreducedforms of FRα. In some embodiments, the antibodies or antigen-bindingfragments are murine IgG, or derivatives thereof. While the antibodiesor antigen-binding fragments may be human, humanized, or chimeric, theantibodies or antigen-binding fragments exemplified herein are murine.In some embodiments, antibodies or antigen-binding fragments may includea light chain CDR1 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 18. In some embodiments, antibodies orantigen-binding fragments may include a light chain CDR2 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 19. Insome embodiments, antibodies or antigen-binding fragments may include alight chain CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 20. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 22. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 23. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 24. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 18; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 19; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 20. Theantibodies or antigen-binding fragments may include a heavy chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 22; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 23; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 24. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 18; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 19; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 20, and also havea heavy chain having a CDR1 amino acid sequence substantially the sameas, or identical to, SEQ ID NO: 22; a CDR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 23; and a CDR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 24.

The described antibodies or antigen-binding fragments may include alight chain variable domain that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 21. In someembodiments, an isolated polynucleotide that includes a sequencesubstantially the same as, or identical to, SEQ ID NO: 53 may encodethis light chain variable domain amino acid sequence. The describedantibodies or antigen-binding fragments may include a heavy chainvariable domain that includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 25. In some embodiments, anisolated polynucleotide that includes a sequence substantially the sameas, or identical to, SEQ ID NO: 57 may encode this heavy chain variabledomain amino acid sequence. The described antibodies or antigen-bindingfragments may include a light and a heavy chain variable domains,wherein the light chain variable domain includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 21, and the heavychain variable domain includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 25. In some embodiments areprovided the 24F12.B1 (24F12) antibody or antigen-binding fragmentsthereof, capable of binding either native or nonreduced forms of FRα.

In some embodiments, the 24F12 antibody is produced byantibody-producing cells deposited with the American Type CultureCollection (10801 University Blvd., Manassas, Va. 20110-2209) on May 19,2011 and have been assigned Accession No. PTA-11886. In someembodiments, the antibodies, or antigen-binding fragments thereof, havethe binding affinity for FRα of the antibodies produced by the depositedantibody-producing cells. In some embodiments, the disclosed antibodies,or antigen-binding fragments thereof, comprise the light and heavy chainCDRs of the antibodies produced by the deposited antibody-producingcells. In some embodiments, the antibodies, or antigen-binding fragmentsthereof, comprise the light and heavy chain variable regions of theantibodies produced by the deposited antibody-producing cells.

Also disclosed are polynucleotides that encode antibodies orantigen-binding fragments that specifically bind to FRα. In someembodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR1 sequencesubstantially the same as, or identical to, SEQ ID NO: 18, for exampleSEQ ID NO: 50. In some embodiments, the isolated polynucleotides encodean antibody or antigen-binding fragment thereof having a light chainCDR2 substantially the same as, or identical to, SEQ ID NO: 19, forexample SEQ ID NO: 51. In some embodiments, the isolated polynucleotidesencode an antibody or antigen-binding fragment thereof having a lightchain CDR3 substantially the same as, or identical to, SEQ ID NO: 20,for example SEQ ID NO: 52. In some embodiments, the isolatedpolynucleotides encode an antibody or antigen-binding fragment thereofhaving a heavy chain CDR1 substantially the same as, or identical to,SEQ ID NO: 22, for example SEQ ID NO: 54. In some embodiments, theisolated polynucleotides encode an antibody or antigen-binding fragmentthereof having a heavy chain CDR2 substantially the same as, oridentical to, SEQ ID NO: 23, for example SEQ ID NO: 55. In someembodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR3 substantiallythe same as, or identical to, SEQ ID NO: 24, for example SEQ ID NO: 56.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a light chain with a CDR1 substantially the same as, oridentical to, SEQ ID NO: 18, for example SEQ ID NO: 50; a CDR2substantially the same as, or identical to, SEQ ID NO: 19, for exampleSEQ ID NO: 51; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 20, for example SEQ ID NO: 52. The polynucleotides may encodean antibody or antigen-binding fragment thereof having a heavy chainCDR1 substantially the same as, or identical to, SEQ ID NO: 22, forexample SEQ ID NO: 54; a CDR2 substantially the same as, or identicalto, SEQ ID NO: 23, for example SEQ ID NO: 55; and a CDR3 substantiallythe same as, or identical to, SEQ ID NO: 24, for example SEQ ID NO: 56.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a light chain CDR1 substantially the same as, oridentical to, SEQ ID NO: 18, for example SEQ ID NO: 50; a CDR2 encodedby a nucleotide sequence substantially the same as, or identical to, SEQID NO: 19, for example SEQ ID NO: 51; and a CDR3 encoded by a nucleotidesequence substantially the same as, or identical to, SEQ ID NO: 20, forexample SEQ ID NO: 52; and a heavy chain CDR1 substantially the same as,or identical to, SEQ ID NO: 22, for example SEQ ID NO: 54; a CDR2substantially the same as, or identical to, SEQ ID NO: 23, for exampleSEQ ID NO: 55; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 24, for example SEQ ID NO: 56. Antigen-binding arrangementsof CDRs may also be engineered using antibody-like proteins as CDRscaffolding. Such engineered antigen-binding proteins are within thescope of the disclosure.

Polynucleotides described herein may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 21, for example SEQ ID NO: 53. In someembodiments the described polynucleotides may encode antibodies orantigen-binding fragments that have a heavy chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 25, for example SEQ ID NO: 57. In someembodiments the described polynucleotides may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 21, for example SEQ ID NO: 53; and a heavychain variable domain segment that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 25, for exampleSEQ ID NO: 57. The polynucleotides capable of encoding the variabledomain segments provided herein may be included on the same, ordifferent, vectors to produce an antibodies or antigen-bindingfragments. Polynucleotides described herein may encode the 24F12antibody or antigen-binding fragments thereof, capable of binding eithernative or nonreduced forms of FRα.

Described herein are isolated antibodies and antigen-binding fragmentsthat specifically bind to FRα in either a native, nonreduced, orchemically preserved form. In some embodiments, the antibodies orantigen-binding fragments are murine IgG, or derivatives thereof. Whilethe antibodies or antigen-binding fragments may be human, humanized, orchimeric, the antibodies or antigen-binding fragments exemplified hereinare murine. In some embodiments, antibodies or antigen-binding fragmentsmay include a light chain CDR1 amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 26. In some embodiments, antibodiesor antigen-binding fragments may include a light chain CDR2 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 27. Insome embodiments, antibodies or antigen-binding fragments may include alight chain CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 28. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 30. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 31. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 32. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 26; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 27; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 28. Theantibodies or antigen-binding fragments may include a heavy chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 30; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 31; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 32. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 26; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 27; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 28, and also havea heavy chain having a CDR1 amino acid sequence substantially the sameas, or identical to, SEQ ID NO: 30; a CDR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 31; and a CDR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 32.

The described antibodies or antigen-binding fragments may include alight chain variable domain that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 29. In someembodiments, an isolated polynucleotide that includes a sequencesubstantially the same as, or identical to, SEQ ID NO: 61 may encodethis light chain variable domain amino acid sequence. The describedantibodies or antigen-binding fragments may include a heavy chainvariable domain that includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 33. In some embodiments, anisolated polynucleotide that includes a sequence substantially the sameas, or identical to, SEQ ID NO: 65 may encode this heavy chain variabledomain amino acid sequence. The described antibodies or antigen-bindingfragments may include a light and a heavy chain variable domains,wherein the light chain variable domain includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 29, and the heavychain variable domain includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 33. In some embodiments areprovided the 26B3.F2 (26B3) antibody or antigen-binding fragmentsthereof, which is capable of binding to the native, nonreduced, orchemically preserved forms of FRα.

In some embodiments, the 26B3 antibody is produced by antibody-producingcells deposited with the American Type Culture Collection (10801University Blvd., Manassas, Va. 20110-2209) on May 19, 2011 and havebeen assigned Accession No. PTA-11885. In some embodiments, theantibodies, or antigen-binding fragments thereof, have the bindingaffinity for FRα of the antibodies produced by the depositedantibody-producing cells. In some embodiments, the disclosed antibodies,or antigen-binding fragments thereof, comprise the light and heavy chainCDRs of the antibodies produced by the deposited antibody-producingcells. In some embodiments, the antibodies, or antigen-binding fragmentsthereof, comprise the light and heavy chain variable regions of theantibodies produced by the deposited antibody-producing cells.

Also disclosed are polynucleotides that encode antibodies orantigen-binding fragments that specifically bind to the native,nonreduced, or chemically preserved forms of FRα. In some embodiments,the isolated polynucleotides encode an antibody or antigen-bindingfragment thereof having a light chain CDR1 sequence substantially thesame as, or identical to, SEQ ID NO: 26, for example SEQ ID NO: 58. Insome embodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR2 substantiallythe same as, or identical to, SEQ ID NO: 27, for example SEQ ID NO: 59.In some embodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR3 substantiallythe same as, or identical to, SEQ ID NO: 28, for example SEQ ID NO: 60.In some embodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR1 substantiallythe same as, or identical to, SEQ ID NO: 30, for example SEQ ID NO: 62.In some embodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR2 substantiallythe same as, or identical to, SEQ ID NO: 31, for example SEQ ID NO: 63.In some embodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR3 substantiallythe same as, or identical to, SEQ ID NO: 32, for example SEQ ID NO: 64.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a light chain with a CDR1 substantially the same as, oridentical to, SEQ ID NO: 26, for example SEQ ID NO: 58; a CDR2substantially the same as, or identical to, SEQ ID NO: 27, for exampleSEQ ID NO: 59; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 28, for example SEQ ID NO: 60. The polynucleotides may encodean antibody or antigen-binding fragment thereof having a heavy chainCDR1 substantially the same as, or identical to, SEQ ID NO: 30, forexample SEQ ID NO: 62; a CDR2 substantially the same as, or identicalto, SEQ ID NO: 31, for example SEQ ID NO: 63; and a CDR3 substantiallythe same as, or identical to, SEQ ID NO: 32, for example SEQ ID NO: 64.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a light chain CDR1 substantially the same as, oridentical to, SEQ ID NO: 26, for example SEQ ID NO: 58; a CDR2 encodedby a nucleotide sequence substantially the same as, or identical to, SEQID NO: 27, for example SEQ ID NO: 59; and a CDR3 encoded by a nucleotidesequence substantially the same as, or identical to, SEQ ID NO: 28, forexample SEQ ID NO: 60; and a heavy chain CDR1 substantially the same as,or identical to, SEQ ID NO: 30, for example SEQ ID NO: 62; a CDR2substantially the same as, or identical to, SEQ ID NO: 31, for exampleSEQ ID NO: 63; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 32, for example SEQ ID NO: 64. Antigen-binding arrangementsof CDRs may also be engineered using antibody-like proteins as CDRscaffolding. Such engineered antigen-binding proteins are within thescope of the disclosure.

Polynucleotides described herein may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 29, for example SEQ ID NO: 61. In someembodiments the described polynucleotides may encode antibodies orantigen-binding fragments that have a heavy chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 33, for example SEQ ID NO: 65. In someembodiments the described polynucleotides may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 29, for example SEQ ID NO: 61; and a heavychain variable domain segment that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 33, for exampleSEQ ID NO: 65. The polynucleotides capable of encoding the variabledomain segments provided herein may be included on the same, ordifferent, vectors to produce an antibodies or antigen-bindingfragments. Polynucleotides described herein may encode the 26B3 antibodyor antigen-binding fragments thereof, capable of binding the native,nonreduced, or chemically preserved forms of FRα.

Vectors comprising the antibody- and antigen-binding fragment-encodingpolynucleotides are provided, as are cells expressing the antibodies orantigen-binding fragments that specifically bind to FRα. Also providedare cells capable of expressing the described vectors. These cells maybe mammalian cells (such as CHO-K1 cells), insect cells (such as Sf7cells), yeast cells, plant cells, or bacteria cells (such as E. coli).The described antibodies may also be produced by hybridoma cells, asdescribed herein.

Methods for Diagnosing Cancer

Provided herein are methods for diagnosing breast, thyroid, colorectal,endometrial, fallopian tube, ovarian or lung cancer of epithelial originin a subject. In some embodiments the described methods involveassessing whether a subject is afflicted with FRα-expressing cancer bydetermining the level of FRα that is present in a sample derived fromthe subject; and comparing the observed level of FRα with the level ofFRα in a control sample, wherein a difference between the level of FRαin the sample derived from the subject and the level of FRα in thecontrol sample is an indication that the subject either is or is notafflicted with an FRα-expressing cancer.

In some embodiments the control sample may be derived from a subjectthat is not afflicted with FRα-expressing cancer. In some embodimentsthe control sample may be derived from a subject that is afflicted withFRα-expressing cancer. In some embodiments where the control sample isderived from a subject that is not afflicted with FRα-expressing cancer,an observed increase in the amount of FRα present in the sample,relative to that observed for the control sample, is an indication thatthe subject being assessed is afflicted with FRα-expressing cancer. Insome embodiments where the control sample is derived from a subject thatis not afflicted with FRα-expressing cancer, an observed decrease orsimilarity in the amount of FRα present in the test sample, relative tothat observed for the control sample, is an indication that the subjectbeing assessed is not afflicted with FRα-expressing cancer. In someembodiments where the control sample is derived from a subject that isafflicted with FRα-expressing cancer, an observed similarity in theamount of FRα present in the test sample, relative to that observed forthe control sample, is an indication that the subject being assessed isafflicted with FRα-expressing cancer. In some embodiments where thecontrol sample is derived from a subject that is afflicted withFRα-expressing cancer, an observed decrease in the amount of FRα presentin the test sample, relative to that observed for the control sample, isan indication that the subject being assessed is not afflicted withFRα-expressing cancer.

In some embodiments the level of FRα in the sample derived from thesubject is assessed by contacting the sample with an antibody that bindsFRα, such as the antibodies described herein. Similar methods may beused to determine if a subject is afflicted with cancer that is notassociated with increased FRα production. The sample assessed for thepresence of FRα may be derived from urine, blood, serum, plasma, saliva,ascites, circulating cells, circulating tumor cells, cells that are nottissue associated (i.e., free cells), tissues (e.g., surgically resectedtumor tissue, biopsies, including fine needle aspiration), histologicalpreparations, and the like.

In some embodiments the described methods involve assessing whether asubject is afflicted with FRα-expressing cancer by determining the levelof FRα associated with a cell or tissue that is present in a samplederived from the subject; and comparing the observed level of FRα withthe level of FRα in a control sample, wherein a difference between thelevel of FRα in the sample derived from the subject and the level of FRαin the control sample is an indication that the subject is afflictedwith an FRα-expressing cancer. In some embodiments the level of FRα inthe sample derived from the subject is assessed by contacting the samplewith an antibody that binds FRα, such as the antibodies describedherein. The sample assessed for the presence of FRα may be circulatingcells, circulating tumor cells, cells that are not tissue associated(i.e., free cells), tissues (e.g., surgically resected tumor tissue,biopsies, including fine needle aspiration), histological preparations,and the like.

In some embodiments the described methods involve assessing whether asubject is afflicted with FRα-expressing cancer by determining the levelof FRα that is not associated with a cell or tissue that is present in asample derived from the subject; and comparing the observed level of FRαwith the level of FRα in a control sample, wherein a difference betweenthe level of FRα in the sample derived from the subject and the level ofFRα in the control sample is an indication that the subject is afflictedwith an FRα-expressing cancer. In some embodiments the level of FRα inthe sample derived from the subject is assessed by contacting the samplewith an antibody that binds FRα, such as the antibodies describedherein. The sample assessed for the presence of FRα may be urine, blood,serum, plasma, saliva, ascites, histological preparations, and the like.

In various embodiments of the described methods, the cancer may beFRα-expressing cancer. In a particular embodiment, the FRα-expressingcancer is ovarian cancer. In some embodiments the FRα-expressing canceris endometrial cancer. In some embodiments the FRα-expressing cancer iscolorectal cancer. In some embodiments the FRα-expressing cancer isbreast cancer. In some embodiments the FRα-expressing cancer is thyroidcancer. In some embodiments the FRα-expressing cancer is fallopian tubecancer. In another embodiment, the FRα-expressing cancer is non-smallcell lung cancer, such as an adenocarcinoma. Alternatively, thedescribed methods may be used to identify cancer that does not expressFRα, such as squamous cell carcinoma. For example, the described methodscould be used to distinguish a FRα-expressing lung cancer, such asadenocarcinoma, from a lung cancer that does not express FRα, such assquamous cell carcinoma. The described methods could be used todistinguish a FRα-expressing breast cancer, such as fibroadenoma, frombreast cancer that does not express FRα, such as cystosarcoma.Furthermore, the described methods could be used to distinguish aFRα-expressing thyroid cancer, such as papillary carcinoma, from thyroidcancer that does not express FRα, such as medullary carcinoma. In someembodiments described herein detection of FRα-expressing cancer cells ina subject may be used to determine that the subject may be treated witha therapeutic agent directed against FRα. In some embodiments thetherapeutic agent directed against FRα may be an antibody, such asFarletuzumab.

In various aspects, the level of FRα is determined by contacting thesample with an antibody, or antigen-binding fragment thereof, that bindsFRα. In some embodiments, the sample may be contacted by more than onetype of antibody, or antigen-binding fragment thereof, that binds FRα.In some embodiments, the sample may be contacted by a first antibody, orantigen-binding fragment thereof, that binds FRα and then contacted by asecond antibody, or antigen-binding fragment thereof, that binds FRα.Antibodies such as those described herein may be used in this capacity.For example, the antibody is selected from the group consisting of:

(a) an antibody, or antigen-binding fragment thereof, that binds thesame epitope as any one of antibody 9F3, antibody 19D4, antibody 24F12,or antibody 26B3;

(b) any one of antibody 9F3, antibody 19D4, antibody 24F12, or antibody26B3, or an antigen-binding fragment thereof;

(c) an antibody, or antigen-binding fragment thereof, that comprises theheavy and light chain CDRs of any one of antibody 9F3, antibody 19D4,antibody 24F12, or antibody 26B3

(d) an antibody, or antigen-binding fragment thereof, that comprises theheavy chain variable domain segment and light chain variable domainsegment of any one of antibody 9F3, antibody 19D4, antibody 24F12, orantibody 26B3, as described in Table 1; or

(e) an antibody having the amino acid sequence of antibody produced byany one of the cell lines deposited with the ATCC having accessionnumber PTA-11887, PTA-11884, PTA-11886, or PTA-11885, or an antigenbinding fragment thereof.

In certain embodiments, the level of FRα is determined by western blotanalysis, radioimmunoassay, immunofluorimetry, immunoprecipitation,equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL)immunoassay, immunohistochemistry, fluorescence-activated cell sorting(FACS) or ELISA assay.

In various embodiments of the foregoing aspects of the invention, thecontrol sample is a standardized control level of FRα in a healthysubject. In other embodiments the control sample may be FRα protein at aknown concentration (e.g., a recombinant or purified FRα proteinsample). In some embodiments, the observed FRα-levels of the testedsubject may be compared with FRα levels observed in samples fromsubjects known to have FRα-expressing cancer or known concentrations ofFRα.

Methods for Monitoring Cancer

Provided herein are methods for monitoring FRα-expressing cancer in asubject. The described methods may be used before treatment for cancer,after treatment for cancer, or both before and after treatment forcancer. In some embodiments the described methods involve assessingwhether FRα-expressing cancer is progressing, regressing, or remainingstable by determining the level of FRα that is present in a test samplederived from the subject; and comparing the observed level of FRα withthe level of FRα in a sample obtained from the subject at an earlierpoint in time, wherein a difference between the level of FRα in the testsample and the earlier sample provides an indication of whether thecancer is progressing, regressing, or remaining stable. In this regard,a test sample with an increased level of FRα, relative to the levelsobserved for the earlier sample, may indicate progression of anFRα-expressing cancer. Conversely, a test sample with a decreased levelof FRα, relative to the levels observed for the earlier sample, mayindicate regression of an FRα-expressing cancer. Accordingly, a testsample with an insignificant difference in the level of FRα, relative tothe levels observed for the earlier sample, may indicate a state ofstable disease for an FRα-expressing cancer. In some embodiments thelevel of FRα in a sample derived from the subject is assessed bycontacting the sample with an antibody that binds FRα, such as theantibodies described herein. The sample assessed for the presence of FRαmay be derived from urine, blood, serum, plasma, saliva, ascites,circulating cells, circulating tumor cells, cells that are not tissueassociated (i.e., free cells), tissues (e.g., surgically resected tumortissue, biopsies, including fine needle aspiration), histologicalpreparations, and the like.

In some embodiments the described methods involve assessing whetherFRα-expressing cancer is progressing, regressing, or remaining stable bydetermining the level of FRαassociated with a cell or tissue that ispresent in a test sample derived from the subject; and comparing theobserved level of FRα with the level of FRα in a sample obtained fromthe subject, in a similar manner, at an earlier point in time, wherein adifference between the level of FRα in the test sample and the earliersample provides an indication of whether the cancer is progressing,regressing, or remaining stable. In this regard, a test sample with anincreased level of FRα, relative to the levels observed for the earliersample, may indicate progression of an FRα-expressing cancer.Conversely, a test sample with a decreased level of FRα, relative to thelevels observed for the earlier sample, may indicate regression of anFRα-expressing cancer. Accordingly, a test sample with an insignificantdifference in the level of FRα, relative to the levels observed for theearlier sample, may indicate a state of stable disease for anFRα-expressing cancer. In some embodiments the level of FRα in a samplederived from the subject is assessed by contacting the sample with anantibody that binds FRα, such as the antibodies described herein. Thesample assessed for the presence of FRα may be circulating cells,circulating tumor cells, cells that are not tissue associated (i.e.,free cells), tissues (e.g., surgically resected tumor tissue, biopsies,including fine needle aspiration), histological preparations, and thelike.

In some embodiments the described methods involve assessing whetherFRα-expressing cancer is progressing, regressing, or remaining stable bydetermining the level of FRα not associated with a cell or tissue thatis present in a test sample derived from the subject; and comparing theobserved level of FRα with the level of FRα in a sample obtained fromthe subject, in a similar manner, at an earlier point in time, wherein adifference between the level of FRα in the test sample and the earliersample provides an indication of whether the cancer is progressing,regressing, or remaining stable. In this regard, a test sample with anincreased level of FRα, relative to the levels observed for the earliersample, may indicate progression of an FRα-expressing cancer.Conversely, a test sample with a decreased level of FRα, relative to thelevels observed for the earlier sample, may indicate regression of anFRα-expressing cancer. Accordingly, a test sample with an insignificantdifference in the level of FRα, relative to the levels observed for theearlier sample, may indicate a state of stable disease for anFRα-expressing cancer. In some embodiments the level of FRα in a samplederived from the subject is assessed by contacting the sample with anantibody that binds FRα, such as the antibodies described herein. Thesample assessed for the presence of FRα may be urine, blood, serum,plasma, saliva, ascites, histological preparations, and the like.

In various embodiments of the described methods, the cancer may beFRα-expressing cancer. In a particular embodiment, the FRα-expressingcancer is ovarian cancer. In some embodiments the FRα-expressing canceris endometrial cancer. In some embodiments the FRα-expressing cancer iscolorectal cancer. In some embodiments the FRα-expressing cancer isbreast cancer. In some embodiments the FRα-expressing cancer is thyroidcancer. In some embodiments the FRα-expressing cancer is fallopian tubecancer. In another embodiment, the FRα-expressing cancer is non-smallcell lung cancer, such as an adenocarcinoma.

In various aspects, the level of FRα is determined by contacting thesample with an antibody, or antigen-binding fragment thereof, that bindsFRα. In some embodiments, the sample may be contacted by more than onetype of antibody, or antigen-binding fragment thereof, that binds FRα.In some embodiments, the sample may be contacted by a first antibody, orantigen-binding fragment thereof, that binds FRα and then contacted by asecond antibody, or antigen-binding fragment thereof, that binds FRα.Antibodies such as those described herein may be used in this capacity.For example, the antibody is selected from the group consisting of:

(a) an antibody, or antigen-binding fragment thereof, that binds thesame epitope as any one of antibody 9F3, antibody 19D4, antibody 24F12,or antibody 26B3;

(b) any one of antibody 9F3, antibody 19D4, antibody 24F12, or antibody26B3, or an antigen-binding fragment thereof;

(c) an antibody, or antigen-binding fragment thereof, that comprises theheavy and light chain CDRs of any one of antibody 9F3, antibody 19D4,antibody 24F12, or antibody 26B3.

In certain embodiments, the level of FRα is determined by western blotanalysis, radioimmunoassay, immunofluorimetry, immunoprecipitation,equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL)immunoassay, immunohistochemistry, fluorescence-activated cell sorting(FACS) or ELISA assay.

Additional aspects of the summarized subject matter are provided ingreater detail in the detailed description and provided examples andassociated figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the migratory patterns of FRα by SDS-PAGE undernonreducing conditions. FRα was assessed in either native (lane 2) orreduced and alkylated (lane 3) form.

FIG. 2 illustrates amino acid residues of FRα (SEQ ID NO:1) thatcomprise the epitopes (shaded regions) for monoclonal antibodies 9F3,24F12, and 26B3, as predicted by hydrogen/deuterium exchange massspectrometry and docking methods.

FIG. 3 shows four western blots of purified recombinant (A) and wholecell lysates (B) from CHO cells expressing FRα or FR homologs FRβ, FRΓor FRΔ were run on SDS-PAGE gels. Proteins were prepared in samplebuffer with or without reducing agents. Panel A, lane 1, molecularweight markers, lanes 2-5. 0.5 μg reduced FRα, FRβ, FRΓ, and FRΔ,respectively; lane 6, blank; lanes 7-10, 0.5 μg nonreduced FRα, FRβ,FRΓ, and FRΔ, respectively. The positive band represents the onlyreactive species in each lane and corresponds to a molecular weight of˜38 kDa. Panel B, lane 1 molecular weight markers, lane 2 CHO-FRα, lane3, CHO-FRβ, lane 4 CHO-FRΔ whole cell lysates prepared in sample bufferwithout reducing agents and fractionated on an SDS-PAGE gel. Each panelis probed with the designated anti-FRα mAb labeled on the right. Themolecular weights for FR are: FRα ˜38 kDA; FRβ ˜30 kDa; FRΓ ˜28 kDa; FRΔ˜26 kDa. The LK26 and BN3.2 antibodies that recognize FRα underdenatured and nonreduced or reduced conditions, respectively, were usedas positive controls.

FIG. 4 shows a formalin-fixed, paraffin-embedded papillary serousovarian cancer tissue sample probed for the presence of FRα withmonoclonal antibody 26B3.

FIG. 5 shows FRα expression in normal tissues. Normal lung (A) andkidney (B) samples stained with antibody 26B3 demonstrate thatexpression of FRα is highly restricted to epithelial cells and has apredominantly apical distribution (images are 20× magnification).

FIG. 6 provides a graphical representation comparing M-scores for lungadenocarcinoma generated using antibody 26B3 or antibody BN3.2.

FIG. 7 shows FRα staining of histologic subtypes of non-small cell lungcarcinoma: (A) lung adenocarcinoma at 20×, (B) lung adenocarcinoma at40×, (C) lung adenosquamous at 20×, and (D) lung squamous cell carcinomaat 40×.

FIG. 8 provides a graphical representation comparing M-scores for lungadenocarcinoma duplicate samples (cores) stained with antibody 26B3.

FIG. 9 illustrates the M-score for FRα distribution of lungadenocarcinoma and squamous cell carcinoma. The mean M-Scores were 19.84(±18.64) and 1.39 (±5.54), respectively (p<0.0001).

FIG. 10 shows FRα expression in three lung adenocarcinoma fine needleaspiration (FNA) samples (A), (B), and (C). Staining of cell blockmaterial from lymph node FNAs with antibody 26B3 demonstrated successfulstaining of FRα, with expression limited to epithelial cells with anapical distribution.

FIG. 11 illustrates the survival functions (death or censor) forsubjects having lung adenocarcinoma who were deemed to be FRα positiveand FRα negative by immunohistochemistry analysis of tissue samplesusing antibody 26B3.

FIG. 12 shows representative tissue microarray (TMA) images stained withantibody 26B3 at either 20× or 40× magnification for (A) ductalcarcinoma in situ, (B)-(D) invasive ductal carcinoma.

FIG. 13 provides a graphical representation of the M-score distribution,as determined by staining with 26B3, relative to the molecular subtype(her-2 (+) and her-2 (−)) of the breast cancer sample.

FIG. 14(A-D) show representative histology samples from stage IV, her2negative breast cancers stained with antibody 26B3 at either 20× or 40×magnification.

FIG. 15 shows representative images of metastatic breast cancer samplesobtained by fine needle aspiration stained with antibody 26B3.

FIG. 16 shows FRα expression in ovarian serous carcinoma. (A) 3+ strong(right field) and 2+ moderate membrane staining (left upper field) arevisible at 10× magnification. (B) Shows the same area as (A) at 20×magnification, confirming a 3+ strong, thick circumferential membranestaining (right field). 2+ moderate membrane staining (left upper field)has a weaker, thinner staining than 3+, and it is circumferential orlocalized to the luminal borders. (C) shows that 1+ weak membranestaining is limited to the luminal borders and requires 40×magnification to visualize. (D) Ovarian surface epithelial cells and theunderlying cortical stromal cells are entirely negative (20×magnification).

FIG. 17 shows FRα expression is limited to the luminal borders normalendometrium with weak 1+ and moderate 2+ intensity at 40× magnification(A). Strong (+3) membrane staining can be observed on the luminalborders of atypical complex hyperplasia at 20× magnification (B).

FIG. 18 shows strong (+3) FRα membrane staining on the luminal bordersof grade 1 adenocarcinoma of endometrium (A). In addition, many tumorcells have 2+ or 3+ cytoplasmic staining (20× magnification). FRαmembrane staining (2+ and 3+) is present on the luminal borders of grade2 adenocarcinoma of endometrium; cytoplasmic staining is weak (20×magnification (B). About 50% of the tumor cells of grade 3adenocarcinoma of endometrium demonstrate 3+ strong, circumferentialmembrane staining with weak cytoplasmic staining at 40× magnification(C).

FIG. 19 shows adenocarcinoma with squamous metaplasia with about 80% ofmetaplastic squamous cells with 2+ and 3+FRα membrane staining and 1+and 2+FRα cytoplasmic staining at 20× magnification (A). Clear cellcarcinoma of endometrium tumor cells have large irregular nuclei,prominent nucleoli and abundant clear cytoplasm. The majority of thesetumor cells have 2+ or 3+FRα membrane staining at 40× magnification (B).

FIG. 20 shows that ciliated and non-ciliated cells of normal fallopiantube have 3+FRα membrane staining on the luminal and lateral cellborders (A). Cytoplasmic staining is also evident (20× magnification).(B) Chronic salpingitis with abundant lymphocytes and plasma cells inthe stroma. Mucosal cells retain 3+FRα staining on the luminal borders(20× magnification). (C) Grade 2 tubal serous adenocarcinoma tumor cellsform complex papillary projections and show 3+FRα membrane staining onthe luminal and lateral cell borders, with cytoplasmic staining alsoevident (20× magnification).

FIG. 21 depicts ovarian cortical serous/tubal cysts. Lining cells reveal3+, strong membrane and cytoplasmic staining (20× magnification).

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The following description characterizes antibodies, and antigen-bindingfragments thereof, that specifically bind to FRα. Also described arerelated polynucleotides capable of encoding these antibodies, andantigen-binding fragments, cells expressing the antibodies, andantigen-binding fragments, as well as associated vectors and detectableantibody labels. In addition, methods of using the antibodies, andantigen-binding fragments, are described. For example, the providedantibodies, and antigen-binding fragments, may be used to diagnoseovarian, breast, thyroid, colorectal, endometrial, fallopian tube, orlung cancer; monitor ovarian, breast, thyroid, colorectal, endometrial,fallopian tube, or lung cancer progression, regression, or stabledisease; to determine whether or not a patient should be treated forcancer, or to determine whether or not a subject is afflicted withFRα-expressing cancer and thus may be amenable to treatment with aFRα-specific anti-cancer therapeutic.

DEFINITIONS

Various terms relating to aspects of the description are used throughoutthe specification and claims. Such terms are to be given their ordinarymeaning in the art unless otherwise indicated. Other specificallydefined terms are to be construed in a manner consistent with thedefinitions provided herein.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of up to ±10% from the specified value, as suchvariations are appropriate to perform the disclosed methods. Unlessotherwise indicated, all numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forthused in the specification and claims are to be understood as beingmodified in all instances by the term “about.” Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thefollowing specification and attached claims are approximations that mayvary depending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the invention are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contain certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements.

“Isolated” means a biological component (such as a nucleic acid, peptideor protein) has been substantially separated, produced apart from, orpurified away from other biological components of the organism in whichthe component naturally occurs, i.e., other chromosomal andextrachromosomal DNA and RNA, and proteins. Nucleic acids, peptides andproteins that have been “isolated” thus include nucleic acids andproteins purified by standard purification methods. “Isolated” nucleicacids, peptides and proteins that can be part of a composition and stillbe isolated if such composition is not part of the native environment ofthe nucleic acid, peptide, or protein. The term also embraces nucleicacids, peptides and proteins prepared by recombinant expression in ahost cell as well as chemically synthesized nucleic acids.

“Polynucleotide,” synonymously referred to as “nucleic acid molecule” or“nucleic acids,” refers to any polyribonucleotide orpolydeoxyribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. “Polynucleotides” include, without limitation single- anddouble-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, “polynucleotide” refers to triple-stranded regions comprisingRNA or DNA or both RNA and DNA. The term polynucleotide also includesDNAs or RNAs containing one or more modified bases and DNAs or RNAs withbackbones modified for stability or for other reasons. “Modified” basesinclude, for example, tritylated bases and unusual bases such asinosine. A variety of modifications may be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically or metabolicallymodified forms of polynucleotides as typically found in nature, as wellas the chemical forms of DNA and RNA characteristic of viruses andcells. “Polynucleotide” also embraces relatively short nucleic acidchains, often referred to as oligonucleotides.

The meaning of “substantially the same” can differ depending on thecontext in which the term is used. Because of the natural sequencevariation likely to exist among heavy and light chains and the genesencoding them, one would expect to find some level of variation withinthe amino acid sequences or the genes encoding the antibodies orantigen-binding fragments described herein, with little or no impact ontheir unique binding properties (e.g., specificity and affinity). Suchan expectation is due in part to the degeneracy of the genetic code, aswell as to the evolutionary success of conservative amino acid sequencevariations, which do not appreciably alter the nature of the encodedprotein. Accordingly, in the context of nucleic acid sequences,“substantially the same” means at least 65% identity between two or moresequences. Preferably, the term refers to at least 70% identity betweentwo or more sequences, more preferably at least 75% identity, morepreferably at least 80% identity, more preferably at least 85% identity,more preferably at least 90% identity, more preferably at least 91%identity, more preferably at least 92% identity, more preferably atleast 93% identity, more preferably at least 94% identity, morepreferably at least 95% identity, more preferably at least 96% identity,more preferably at least 97% identity, more preferably at least 98%identity, and more preferably at least 99% or greater identity. Suchidentity may be determined using nBLAST algorithm (Altschul et al.,(1990) Proc. Natl. Acad. Sci. USA 87:2264-8; Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-7).

The degree of variation that may occur within the amino acid sequence ofa protein without having a substantial effect on protein function ismuch lower than that of a nucleic acid sequence, since the samedegeneracy principles do not apply to amino acid sequences. Accordingly,in the context of an antibody or antigen-binding fragment,“substantially the same” means antibodies or antigen-binding fragmentshaving 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity tothe antibodies or antigen-binding fragments described. Other embodimentsinclude FRα specific antibodies, or antigen-binding fragments, that haveframework, scaffold, or other non-binding regions that do not sharesignificant identity with the antibodies and antigen-binding fragmentsdescribed herein, but do incorporate one or more CDRs or other sequencesneeded to confer binding that are 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to such sequences described herein.

A “vector” is a replicon, such as plasmid, phage, cosmid, or virus inwhich another nucleic acid segment may be operably inserted so as tobring about the replication or expression of the segment.

A cell has been “transformed” when exogenous or heterologous nucleicacids such as DNA have been introduced inside the cell. The transformingDNA may or may not be integrated (covalently linked) into the genome ofthe cell. In prokaryotes, yeast, and mammalian cells for example, thetransforming DNA may be maintained on an episomal element such as aplasmid. With respect to eukaryotic cells, a stably transformed cell, or“stable cell” is demonstrated by the ability of the eukaryotic cell toestablish cell lines or clones comprised of a population of daughtercells containing the transforming DNA. A “clone” is a population ofcells derived from a single cell or common ancestor by mitosis. A “cellline” is a clone of a primary cell that is capable of stable growth invitro for many generations. In some examples provided herein, cells aretransformed by transfecting the cells with DNA.

The terms “express” and “produce” are used synonymously herein, andrefer to the biosynthesis of a gene product. These terms encompass thetranscription of a gene into RNA. These terms also encompass translationof RNA into one or more polypeptides, and further encompass allnaturally occurring post-transcriptional and post-translationalmodifications. The expression or production of an antibody orantigen-binding fragment thereof may be within the cytoplasm of thecell, or into the extracellular milieu such as the growth medium of acell culture.

The terms “treating” or “treatment” refer to any success or indicia ofsuccess in the attenuation or amelioration of an injury, pathology orcondition, including any objective or subjective parameter such asabatement, remission, diminishing of symptoms or making the conditionmore tolerable to the patient, slowing in the rate of degeneration ordecline, making the final point of degeneration less debilitating,improving a subject's physical or mental well-being, or prolonging thelength of survival. The treatment may be assessed by objective orsubjective parameters; including the results of a physical examination,neurological examination, or psychiatric evaluations.

“Antibody” refers to all isotypes of immunoglobulins (IgG, IgA, IgE,IgM, IgD, and IgY) including various monomeric and polymeric forms ofeach isotype, unless otherwise specified.

Antigen-binding fragments are any proteinaceous structure that mayexhibit binding affinity for a particular antigen. Some antigen-bindingfragments are composed of portions of intact antibodies that retainantigen-binding specificity of the parent antibody molecule. Forexample, antigen-binding fragments may comprise at least one variableregion (either a heavy chain or light chain variable region) or one ormore CDRs of an antibody known to bind a particular antigen. Examples ofsuitable antigen-binding fragments include, without limitation diabodiesand single-chain molecules as well as Fab, F(ab′)2, Fc, Fabc, and Fvmolecules, single chain (Sc) antibodies, individual antibody lightchains, individual antibody heavy chains, chimeric fusions betweenantibody chains or CDRs and other proteins, protein scaffolds, heavychain monomers or dimers, light chain monomers or dimers, dimersconsisting of one heavy and one light chain, and the like. All antibodyisotypes may be used to produce antigen-binding fragments. Additionally,antigen-binding fragments may include non-antibody proteinaceousframeworks that may successfully incorporate polypeptide segments in anorientation that confers affinity for a given antigen of interest, suchas protein scaffolds. Antigen-binding fragments may be recombinantlyproduced or produced by enzymatic or chemical cleavage of intactantibodies. The phrase “an antibody or antigen-binding fragment thereof”may be used to denote that a given antigen-binding fragment incorporatesone or more amino acid segments of the antibody referred to in thephrase.

“Specific binding” when used in the context of antibodies, or antibodyfragments, represents binding via domains encoded by immunoglobulingenes or fragments of immunoglobulin genes to one or more epitopes of aprotein of interest, but which do not substantially recognize and bindother molecules in a sample containing a mixed population of antigenicmolecules. Typically, an antibody binds to a cognate antigen with a Kdof less than about 1×10-8 M, as measured by a surface plasmon resonanceassay or a cell binding assay.

The term “subject” refers to human and non-human animals, including allvertebrates, e.g., mammals and non-mammals, such as non-human primates,mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians,and reptiles. In many embodiments of the described methods, the subjectis a human.

As used herein, the term “folate receptor alpha” (also referred to asFRα, FR-alpha, FOLR-1 or FOLR1) refers to the alpha isoform of the highaffinity receptor for folate. Membrane bound FRα is attached to the cellsurface by a glycosyl phosphatidylinositol (GPI) anchor. Soluble formsof FRα may be derived by the action of proteases or phospholipase onmembrane anchored folate receptors. The amino acid sequence for humanFRα is set forth herein as SEQ ID NO: 1. Variants, for example,naturally occurring allelic variants or sequences containing at leastone amino acid substitution, are encompassed by the terms as usedherein. As will be appreciated by those skilled in the art, cellassociated and non-cell associated forms of human FRα may encompassvariant forms of SEQ ID NO:1.

The term “sample” as used herein refers to a collection of similarfluids, cells, or tissues (e.g., surgically resected tumor tissue,biopsies, including fine needle aspiration), isolated from a subject, aswell as fluids, cells, or tissues present within a subject. In someembodiments the sample is a biological fluid. Biological fluids aretypically liquids at physiological temperatures and may includenaturally occurring fluids present in, withdrawn from, expressed orotherwise extracted from a subject or biological source. Certainbiological fluids derive from particular tissues, organs or localizedregions and certain other biological fluids may be more globally orsystemically situated in a subject or biological source. Examples ofbiological fluids include blood, serum and serosal fluids, plasma,lymph, urine, saliva, cystic fluid, tear drops, feces, sputum, mucosalsecretions of the secretory tissues and organs, vaginal secretions,ascites fluids such as those associated with non-solid tumors, fluids ofthe pleural, pericardial, peritoneal, abdominal and other body cavities,fluids collected by bronchial lavage and the like.

Biological fluids may also include liquid solutions contacted with asubject or biological source, for example, cell and organ culture mediumincluding cell or organ conditioned medium, lavage fluids and the like.The term “sample,” as used herein, encompasses materials removed from asubject or materials present in a subject.

The term “progression,” as used in the context of progression of anFRα-expressing cancer, includes the change of a cancer from a lesssevere to a more severe state. This could include an increase in thenumber or severity of tumors, the degree of metastasis, the speed withwhich the cancer is growing or spreading, and the like. For example,“the progression of ovarian cancer” includes the progression of such acancer from a less severe to a more severe state, such as theprogression from stage I to stage II, from stage II to stage III, etc.

The term “regression,” as used in the context of regression of anFRα-expressing cancer, includes the change of a cancer from a moresevere to a less severe state. This could include a decrease in thenumber or severity of tumors, the degree of metastasis, the speed withwhich the cancer is growing or spreading, and the like. For example,“the regression of ovarian cancer” includes the regression of such acancer from a more severe to a less severe state, such as theprogression from stage III to stage II, from stage II to stage I, etc.

The term “stable” as used in the context of stable FRα-expressingcancer, is intended to describe a disease condition that is not, or hasnot, changed significantly enough over a clinically relevant period oftime to be considered a progressing cancer or a regressing cancer.

The embodiments described herein are not limited to particular methods,reagents, compounds, compositions or biological systems, which can, ofcourse, vary.

FRα-Specific Antibodies and Antigen-Binding Fragments

Described herein are isolated monoclonal antibodies or antigen-bindingfragments that specifically bind FRα. The general structure of anantibody molecule comprises an antigen binding domain, which includesheavy and light chains, and the Fc domain, which serves a variety offunctions, including complement fixation and binding antibody receptors.

The described antibodies or antigen-binding fragments include allisotypes, IgA, IgD, IgE, IgG and IgM, and synthetic multimers of thefour-chain immunoglobulin structure. The described antibodies orantigen-binding fragments also include the IgY isotype generally foundin hen or turkey serum and hen or turkey egg yolk.

The antibodies or antigen-binding fragments disclosed in the examplessection are derived from mice. Similar antibodies may be derived fromany species by recombinant means. For example, the antibodies orantigen-binding fragments may be chimeric rat, goat, horse, swine,bovine, chicken, rabbit, camelid, donkey, human, and the like. For usein administration to humans, non-human derived antibodies orantigen-binding fragments may be genetically or structurally altered tobe less antigenic upon administration to a human patient.

In some embodiments, the antibodies or antigen-binding fragments arechimeric. As used herein, the term “chimeric” refers to an antibody, orantigen-binding fragment thereof, having at least some portion of atleast one variable domain derived from the antibody amino acid sequenceof a non-human mammal, a rodent, or a reptile, while the remainingportions of the antibody, or antigen-binding fragment thereof, arederived from a human. For example, a chimeric antibody may comprise amouse antigen binding domain with a human Fc or other such structuraldomain.

In some embodiments, the antibodies are humanized antibodies. Humanizedantibodies may be chimeric immunoglobulins, immunoglobulin chains orfragments thereof (such as Fv, Fab, Fab′, F(ab′)2 or otherantigen-binding subsequences of antibodies) that contain minimalsequence derived from non-human immunoglobulin. For the most part,humanized antibodies are human immunoglobulins (recipient antibody) inwhich residues from a complementary-determining region (CDR) of therecipient are replaced by residues from a CDR of a non-human species(donor antibody) such as mouse, rat or rabbit having the desiredspecificity, affinity, and capacity. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the framework regions are those of a humanimmunoglobulin sequence. The humanized antibody may include at least aportion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin.

The antibodies or antigen-binding fragments described herein can occurin a variety of forms, but will include one or more of the antibodyvariable domain segments or CDRs shown in Table 1. The isotypes of theantibodies described in Table 1 are shown in parentheses to describe theconstant region of each antibody, which are known to have conservedsequences.

TABLE 1 Antibody segments of the described antibodies and antigen-binding fragmentsthereof (“Lc” denotes light chain and “Hc” denotes heavy chain). SEQAntibody Segment ID NO. SequenceMonoclonal antibody 9F3 (murine IgG2a constant region) Lc CDR1 2RASSTVSYSYLH Lc CDR2 3 GTSNLAS Lc CDR3 4 QQYSGYPLT Lc variable domain 5PAIMSASPGEKVTMTCRASSTVSYSYLHWYQQ segmentKSGASPQLWIYGTSNLASGVPARFSGSGSGTSY SLTISSVEAEDAATYYCQQYSGYPLTFGAGTKLELKRADAAP Hc CDR1 6 SGYYWN Hc CDR2 7 YIKSDGSNNYNPSLKN Hc CDR3 8 EWKAMDYHc variable domain 9 ESGPGLVRPSQSLSLTCSVTGYSITSGYYWNWIR segmentQFPGSRLEWMGYIKSDGSNNYNPSLKNRISITR DTSKNQFFLKLNSVTTEDTATYFCTREWKAMDYWGQGTSVTVSSAKTTPPSVYPLAPGCGDTMonoclonal antibody 19D4 (murine IgG2a contstant region) Lc CDR1 10RASESVDTYGNNFIH Lc CDR2 11 LASNLES Lc CDR3 12 QQNNGDPWTLc variable domain 13 PASLAVSLGQRATISCRASESVDTYGNNFIHWY segmentQQKPGQPPKLLIYLASNLESGVPARFSGSGSRTD FTLTIDPVEADDAATYYCQQNNGDPWTFGGGTKLEIKRADAAP Hc CDR1 14 HPYMH Hc CDR2 15 RIDPANGNTKYDPKFQG Hc CDR3 16EEVADYTMDY Hc variable domain 17 GAELVKPGASVKLSCTASGFNIKHPYMHWVKQsegment RPDQGLEWIGRIDPANGNTKYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCGREEVADYT MDYWGQGTSVTVSSAKTTAPSVYPLAPVMonoclonal antibody 24F12 (murine IgG1 contstant region) Lc CDR1 18SASQGINNFLN Lc CDR2 19 YTSSLHS Lc CDR3 20 QHFSKLPWT Lc variable domain21 TSSLSASLGDRVTISCSASQGINNFLNWYQQKP DGTVKLLIYYTSSLHSGVPSRFSGSGSGTDYSLTISNLEPEDIAIYYCQHFSKLPWTFGGGTKLEIKR ADAAP Hc CDR1 22 SYAMS Hc CDR2 23EIGSGGSYTYYPDTVTG Hc CDR3 24 ETTAGYFDY Hc variable domain 25SGGGLVRPGGSLKLSCAASGFTFSSYAMSWVR QSPEKRLEWVAEIGSGGSYTYYPDTVTGRFTISRDNAKSTLYLEMSSLRSEDTAIYYCARETTAGYF DYWGQGTTLTVSSMonoclonal antibody 26B3 (murine IgG1 contstant region) Lc CDR1 26RTSENIFSYLA Lc CDR2 27 NAKTLAE Lc CDR3 28 QHHYAFPWT Lc variable domain29 PASLSASVGETVTITCRTSENIFSYLAWYQQKQ segmentGISPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLK INSLQPEDFGSYYCQHHYAFPWTFGGGSKLEIKRADAAP Hc CDR1 30 GYFMN Hc CDR2 31 RIFPYNGDTFYNQKFKG Hc CDR3 32 GTHYFDYHc variable domain 33 GPELVKPGASVKISCKASDYSFTGYFMNWVMQ segmentSHGKSLEWIGRIFPYNGDTFYNQKFKGRATLTV DKSSSTAHMELRSLASEDSAVYFCARGTHYFDYWGQGTTLTVSSAKTTPPSVYPLAPGSAAQTMonoclonal antibody 9F3 (murine IgG2a contstant region) Lc CDR1 34AGGGCCAGCTCAACTGTAAGTTACAGTTACTT GCAC Lc CDR2 35 GGCACATCCAACTTGGCTTCTLc CDR3 36 CAGCAGTACAGTGGTTACCCACTCACG Lc variable domain 37CCAGCAATCATGTCTGCATCTCCAGGGGAAA segment AGGTCACCATGACCTGCAGGGCCAGCTCAACTGTAAGTTACAGTTACTTGCACTGGTACCAGC AGAAGTCAGGTGCCTCCCCCCAACTCTGGATTTATGGCACATCCAACTTGGCTTCTGGAGTCCC TGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGTGTGGAGGCT GAAGATGCTGCCACTTATTACTGCCAGCAGTACAGTGGTTACCCACTCACGTTCGGTGCTGGGA CCAAGCTGGAGCTGAAACGGGCTGATGCTGC ACCAACHc CDR1 38 AGTGGTTATTACTGGAAC Hc CDR2 39 TACATAAAGTCCGACGGTAGCAATAATTACAACCCATCTCTCAAAAAT Hc CDR3 40 GAGTGGAAGGCTATGGACTAC Hc variable domain 41GAGTCAGGACCTGGCCTCGTGAGACCTTCTCA segmentGTCTCTGTCTCTCACCTGCTCTGTCACTGGCT ACTCCATCACCAGTGGTTATTACTGGAACTGGATCCGGCAGTTTCCAGGAAGCAGACTGGAAT GGATGGGCTACATAAAGTCCGACGGTAGCAATAATTACAACCCATCTCTCAAAAATCGAATCT CCATCACTCGTGACACATCTAAGAACCAGTTTTTCCTGAAGTTGAATTCTGTGACTACTGAGGA CACAGCTACATATTTCTGTACAAGGGAGTGGAAGGCTATGGACTACTGGGGTCAGGGAACCT CAGTCACCGTCTCCTCAGCCAAAACAACACCCCCATCAGTCTATCCACTGGCCCCTGGGTGTG GAGATACAACMonoclonal antibody 19D4 (murine IgG2a contstant region) Lc CDR1 42AGAGCCAGTGAAAGTGTTGATACTTATGGCA ATAATTTTATACAC Lc CDR2 43CTTGCATCCAACCTAGAATCT Lc CDR3 44 CAGCAAAATAATGGGGATCCGTGGACGLc variable domain 45 CCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAG segmentGGCCACCATATCCTGCAGAGCCAGTGAAAGT GTTGATACTTATGGCAATAATTTTATACACTGGTACCAGCAGAAACCAGGACAGCCACCCAAA CTCCTCATTTATCTTGCATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGG TCTAGGACAGACTTCACCCTCACCATTGATCCTGTGGAGGCTGATGATGCTGCAACCTATTACT GTCAGCAAAATAATGGGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAGATCAAACGG GCTGATGCTGCACCAA Hc CDR1 46CACCCCTATATGCAC Hc CDR2 47 AGGATTGATCCTGCGAATGGTAATACTAAATATGACCCGAAGTTCCAGGGC Hc CDR3 48 GAGGAGGTGGCGGACTATACTATGGACTACHc variable domain 49 GGGGCAGAGCTTGTGAAGCCAGGGGCCTCAG segmentTCAAGTTGTCCTGCACAGCTTCTGGCTTCAAC ATTAAACACCCCTATATGCACTGGGTGAAGCAGAGGCCTGACCAGGGCCTGGAGTGGATTGG AAGGATTGATCCTGCGAATGGTAATACTAAATATGACCCGAAGTTCCAGGGCAAGGCCACTA TAACAGCAGACACATCCTCCAACACAGCCTACCTACAGCTCAGCAGCCTGACATCTGAGGAC ACTGCCGTCTATTACTGTGGTAGAGAGGAGGTGGCGGACTATACTATGGACTACTGGGGTCA AGGAACCTCAGTCACCGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCC TGTGTGMonoclonal antibody 24F12 (murine IgG1 contstant region) Lc CDR1 50AGTGCAAGTCAGGGCATTAACAATTTTTTAAAC Lc CDR2 51 TACACATCAAGTTTACACTCALc CDR3 52 CAGCACTTTAGTAAGCTTCCGTGGACG Lc variable domain 53ACATCCTCCCTGTCTGCCTCTCTGGGAGACAG segment AGTCACCATCAGTTGCAGTGCAAGTCAGGGCATTAACAATTTTTTAAACTGGTATCAGCAGAA ACCAGATGGCACTGTTAAACTCCTGATCTATTACACATCAAGTTTACACTCAGGAGTCCCATCA AGGTTCAGTGGCAGTGGGTCTGGGACAGATTATTCTCTCACCATCAGCAACCTGGAACCTGAA GATATTGCCATATACTATTGTCAGCACTTTAGTAAGCTTCCGTGGACGTTCGGTGGAGGCACC AAGCTGGAAATCAAACGGGCTGATGCTGCAC CAACHc CDR1 54 AGCTATGCCATGTCT Hc CDR2 55 GAAATTGGTAGTGGTGGTAGTTACACCTACTATCCAGACACTGTGACGGGC Hc CDR3 56 GAAACTACGGCGGGCTACTTTGACTACHc variable domain 57 TCTGGGGGAGGCTTAGTGAGGCCTGGAGGGT segmentCCCTGAAACTCTCCTGTGCAGCCTCTGGATTC ACTTTCAGTAGCTATGCCATGTCTTGGGTTCGCCAGTCTCCAGAGAAGAGGCTGGAGTGGGTC GCAGAAATTGGTAGTGGTGGTAGTTACACCTACTATCCAGACACTGTGACGGGCCGATTCAC CATCTCCAGAGACAATGCCAAGAGCACCCTGTACCTGGAAATGAGCAGTCTGAGGTCTGAGG ACACGGCCATCTATTACTGTGCAAGGGAAACTACGGCGGGCTACTTTGACTACTGGGGCCAA GGCACCACTCTCACAGTCTCCTCAMonoclonal antibody 26B3 (murine IgG1 contstant region) Lc CDR1 58CGAACAAGTGAGAATATTTTCAGTTATTTAGCA Lc CDR2 59 AATGCAAAAACCTTAGCAGAGLc CDR3 60 CAACATCATTATGCTTTTCCGTGGACG Lc variable domain 61CCAGCCTCCCTATCTGCATCTGTGGGAGAAAC segment TGTCACCATCACATGTCGAACAAGTGAGAATATTTTCAGTTATTTAGCATGGTATCAGCAGAA ACAGGGAATATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGAGGGTGTGCCATC AAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGA AGATTTTGGGAGTTATTACTGTCAACATCATTATGCTTTTCCGTGGACGTTCGGTGGAGGCTCC AAGCTGGAAATCAAACGGGCTGATGCTGCAC CAACHc CDR1 62 GGCTACTTTATGAAC Hc CDR2 63 CGTATTTTTCCTTACAATGGTGATACTTTCTACAACCAGAAGTTCAAGGGC Hc CDR3 64 GGGACTCATTACTTTGACTAC Hc variable domain65 GGACCTGAGCTGGTGAAGCCTGGGGCTTCAG segmentTGAAGATATCCTGCAAGGCTTCTGATTACTCT TTTACTGGCTACTTTATGAACTGGGTGATGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGA CGTATTTTTCCTTACAATGGTGATACTTTCTACAACCAGAAGTTCAAGGGCAGGGCCACATTGA CTGTAGACAAATCCTCTAGCACAGCCCACATGGAGCTCCGGAGCCTGGCATCTGAGGACTCT GCAGTCTATTTTTGTGCAAGAGGGACTCATTACTTTGACTACTGGGGCCAAGGCACCACTCTCA CTGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCA AACTAA

Described herein are isolated antibodies and antigen-binding fragmentsthat specifically bind to FRα. In some embodiments, the antibodies orantigen-binding fragments are murine IgG, or derivatives thereof. Whilethe antibodies or antigen-binding fragments may be human, humanized, orchimeric, the antibodies or antigen-binding fragments exemplified hereinare murine. In some embodiments, antibodies or antigen-binding fragmentsmay include a light chain CDR1 amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 2. In some embodiments, antibodiesor antigen-binding fragments may include a light chain CDR2 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 3. Insome embodiments, antibodies or antigen-binding fragments may include alight chain CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 4. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 6. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 7. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 8. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 2; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 3; and a CDR3 amino acid sequence substantiallythe same as, or identical to, SEQ ID NO: 4. The antibodies orantigen-binding fragments may include a heavy chain having a CDR1 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 6;a CDR2 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 7; and a CDR3 amino acid sequence substantially the same as,or identical to, SEQ ID NO: 8. The antibodies or antigen-bindingfragments may include a light chain having a CDR1 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 2; a CDR2 aminoacid sequence substantially the same as, or identical to, SEQ ID NO: 3;and a CDR3 amino acid sequence substantially the same as, or identicalto, SEQ ID NO: 4, and also have a heavy chain having a CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 6; aCDR2 amino acid sequence substantially the same as, or identical to, SEQID NO: 7; and a CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 8.

The described antibodies or antigen-binding fragments may include alight chain variable domain that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 5. In someembodiments, an isolated polynucleotide that includes a sequencesubstantially the same as, or identical to, SEQ ID NO: 37 may encodethis light chain variable domain amino acid sequence. The describedantibodies or antigen-binding fragments may include a heavy chainvariable domain that includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 9. In some embodiments, an isolatedpolynucleotide that includes a sequence substantially the same as, oridentical to, SEQ ID NO: 41 may encode this heavy chain variable domainamino acid sequence. The described antibodies or antigen-bindingfragments may include a light and a heavy chain variable domains,wherein the light chain variable domain includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 5, and the heavychain variable domain includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 9.

In some embodiments, the antibodies are produced by antibody-producingcells deposited with the American Type Culture Collection (10801University Blvd., Manassas, Va. 20110-2209) on May 19, 2011 and havebeen assigned Accession No. PTA-11887. In some embodiments, theantibodies, or antigen-binding fragments thereof, have the bindingaffinity for FRα of the antibodies produced by the depositedantibody-producing cells. In some embodiments, the disclosed antibodies,or antigen-binding fragments thereof, comprise the light and heavy chainCDRs of the antibodies produced by the deposited antibody-producingcells. In some embodiments, the antibodies, or antigen-binding fragmentsthereof, comprise the light and heavy chain variable regions of theantibodies produced by the deposited antibody-producing cells.

Also disclosed are isolated polynucleotides that encode antibodies orantigen-binding fragments that specifically bind to FRα. In someembodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR1 sequencesubstantially the same as, or identical to, SEQ ID NO: 2, for exampleSEQ ID NO: 34. In some embodiments, the isolated polynucleotides encodean antibody or antigen-binding fragment thereof having a light chainCDR2 substantially the same as, or identical to, SEQ ID NO: 3, forexample SEQ ID NO: 35. In some embodiments, the isolated polynucleotidesencode an antibody or antigen-binding fragment thereof having a lightchain CDR3 substantially the same as, or identical to, SEQ ID NO: 4, forexample SEQ ID NO: 36. In some embodiments, the isolated polynucleotidesencode an antibody or antigen-binding fragment thereof having a heavychain CDR1 substantially the same as, or identical to, SEQ ID NO: 6, forexample SEQ ID NO: 38. In some embodiments, the isolated polynucleotidesencode an antibody or antigen-binding fragment thereof having a heavychain CDR2 substantially the same as, or identical to, SEQ ID NO: 7, forexample SEQ ID NO: 39. In some embodiments, the isolated polynucleotidesencode an antibody or antigen-binding fragment thereof having a heavychain CDR3 substantially the same as, or identical to, SEQ ID NO: 8, forexample SEQ ID NO: 40. The isolated polynucleotides may encode anantibody or antigen-binding fragment thereof having a light chain with aCDR1 substantially the same as, or identical to, SEQ ID NO: 2, forexample SEQ ID NO: 34; a CDR2 substantially the same as, or identicalto, SEQ ID NO: 3, for example SEQ ID NO: 35; and a CDR3 substantiallythe same as, or identical to, SEQ ID NO: 4, for example SEQ ID NO: 36.The isolated polynucleotides may encode an antibody or antigen-bindingfragment thereof having a heavy chain CDR1 substantially the same as, oridentical to, SEQ ID NO: 6, for example SEQ ID NO: 38; a CDR2substantially the same as, or identical to, SEQ ID NO: 7, for exampleSEQ ID NO: 39; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 8, for example SEQ ID NO: 40. The isolated polynucleotidesmay encode an antibody or antigen-binding fragment thereof having alight chain CDR1 substantially the same as, or identical to, SEQ ID NO:2, for example SEQ ID NO: 34; a CDR2 encoded by a nucleotide sequencesubstantially the same as, or identical to, SEQ ID NO: 3, for exampleSEQ ID NO: 35; and a CDR3 encoded by a nucleotide sequence substantiallythe same as, or identical to, SEQ ID NO: 4, for example SEQ ID NO: 36;and a heavy chain CDR1 substantially the same as, or identical to, SEQID NO: 6, for example SEQ ID NO: 38; a CDR2 substantially the same as,or identical to, SEQ ID NO: 7, for example SEQ ID NO: 39; and a CDR3substantially the same as, or identical to, SEQ ID NO: 8, for exampleSEQ ID NO: 40. Antigen-binding arrangements of CDRs may also beengineered using antibody-like proteins as CDR scaffolding. Suchengineered antigen-binding proteins are within the scope of thedisclosure.

Polynucleotides described herein may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 5, for example SEQ ID NO: 37. In someembodiments the described isolated polynucleotides may encode antibodiesor antigen-binding fragments that have a heavy chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 9, for example SEQ ID NO: 41. In someembodiments the described isolated polynucleotides may encode antibodiesor antigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 5, for example SEQ ID NO: 37; and a heavychain variable domain segment that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 9, for exampleSEQ ID NO: 41. The isolated polynucleotides capable of encoding thevariable domain segments provided herein may be included on the same, ordifferent, vectors to produce antibodies or antigen-binding fragments.

Described herein are isolated antibodies and antigen-binding fragmentsthat specifically bind to FRα. In some embodiments, the antibodies orantigen-binding fragments are murine IgG, or derivatives thereof. Whilethe antibodies or antigen-binding fragments may be human, humanized, orchimeric, the antibodies or antigen-binding fragments exemplified hereinare murine. In some embodiments, antibodies or antigen-binding fragmentsmay include a light chain CDR1 amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 10. In some embodiments, antibodiesor antigen-binding fragments may include a light chain CDR2 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 11. Insome embodiments, antibodies or antigen-binding fragments may include alight chain CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 12. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 14. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 15. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 16. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 10; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 11; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 12. Theantibodies or antigen-binding fragments may include a heavy chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 14; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 15; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 16. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 10; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 11; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 12, and also havea heavy chain having a CDR1 amino acid sequence substantially the sameas, or identical to, SEQ ID NO: 14; a CDR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 15; and a CDR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 16.

The described antibodies or antigen-binding fragments may include alight chain variable domain that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 13. In someembodiments, an isolated polynucleotide that includes a sequencesubstantially the same as, or identical to, SEQ ID NO: 45 may encodethis light chain variable domain amino acid sequence. The describedantibodies or antigen-binding fragments may include a heavy chainvariable domain that includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 17. In some embodiments, anisolated polynucleotide that includes a sequence substantially the sameas, or identical to, SEQ ID NO: 49 may encode this heavy chain variabledomain amino acid sequence. The described antibodies or antigen-bindingfragments may include a light and a heavy chain variable domains,wherein the light chain variable domain includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 13, and the heavychain variable domain includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 17.

In some embodiments, the antibodies are produced by antibody-producingcells deposited with the American Type Culture Collection (10801University Blvd., Manassas, Va. 20110-2209) on May 19, 2011 and havebeen assigned Accession No. PTA-11884. In some embodiments, theantibodies, or antigen-binding fragments thereof, have the bindingaffinity for FRα of the antibodies produced by the depositedantibody-producing cells. In some embodiments, the disclosed antibodies,or antigen-binding fragments thereof, comprise the light and heavy chainCDRs of the antibodies produced by the deposited antibody-producingcells. In some embodiments, the antibodies, or antigen-binding fragmentsthereof, comprise the light and heavy chain variable regions of theantibodies produced by the deposited antibody-producing cells.

Also disclosed are isolated polynucleotides that encode antibodies orantigen-binding fragments that specifically bind to FRα. In someembodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR1 sequencesubstantially the same as, or identical to, SEQ ID NO: 10, for exampleSEQ ID NO: 42. In some embodiments, the isolated polynucleotides encodean antibody or antigen-binding fragment thereof having a light chainCDR2 substantially the same as, or identical to, SEQ ID NO: 11, forexample SEQ ID NO: 43. In some embodiments, the isolated polynucleotidesencode an antibody or antigen-binding fragment thereof having a lightchain CDR3 substantially the same as, or identical to, SEQ ID NO: 12,for example SEQ ID NO: 44. In some embodiments, the isolatedpolynucleotides encode an antibody or antigen-binding fragment thereofhaving a heavy chain CDR1 substantially the same as, or identical to,SEQ ID NO: 14, for example SEQ ID NO: 46. In some embodiments, theisolated polynucleotides encode an antibody or antigen-binding fragmentthereof having a heavy chain CDR2 substantially the same as, oridentical to, SEQ ID NO: 15, for example SEQ ID NO: 47. In someembodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR3 substantiallythe same as, or identical to, SEQ ID NO: 16, for example SEQ ID NO: 48.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a light chain with a CDR1 substantially the same as, oridentical to, SEQ ID NO: 10, for example SEQ ID NO: 42; a CDR2substantially the same as, or identical to, SEQ ID NO: 11, for exampleSEQ ID NO: 43; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 12, for example SEQ ID NO: 44. The polynucleotides may encodean antibody or antigen-binding fragment thereof having a heavy chainCDR1 substantially the same as, or identical to, SEQ ID NO: 14, forexample SEQ ID NO: 46; a CDR2 substantially the same as, or identicalto, SEQ ID NO: 15, for example SEQ ID NO: 47; and a CDR3 substantiallythe same as, or identical to, SEQ ID NO: 16, for example SEQ ID NO: 48.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a light chain CDR1 substantially the same as, oridentical to, SEQ ID NO: 10, for example SEQ ID NO: 42; a CDR2 encodedby a nucleotide sequence substantially the same as, or identical to, SEQID NO: 11, for example SEQ ID NO: 43; and a CDR3 encoded by a nucleotidesequence substantially the same as, or identical to, SEQ ID NO: 12, forexample SEQ ID NO: 44; and a heavy chain CDR1 substantially the same as,or identical to, SEQ ID NO: 14, for example SEQ ID NO: 46; a CDR2substantially the same as, or identical to, SEQ ID NO: 15, for exampleSEQ ID NO: 47; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 16, for example SEQ ID NO: 48. Antigen-binding arrangementsof CDRs may also be engineered using antibody-like proteins as CDRscaffolding. Such engineered antigen-binding proteins are within thescope of the disclosure.

Polynucleotides described herein may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 13, for example SEQ ID NO: 45. In someembodiments the described polynucleotides may encode antibodies orantigen-binding fragments that have a heavy chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 17, for example SEQ ID NO: 49. In someembodiments the described polynucleotides may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 13, for example SEQ ID NO: 45; and a heavychain variable domain segment that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 17, for exampleSEQ ID NO: 49. The polynucleotides capable of encoding the variabledomain segments provided herein may be included on the same, ordifferent, vectors to produce antibodies or antigen-binding fragments.

Described herein are isolated antibodies and antigen-binding fragmentsthat specifically bind to FRα. In some embodiments, the antibodies orantigen-binding fragments are murine IgG, or derivatives thereof. Whilethe antibodies or antigen-binding fragments may be human, humanized, orchimeric, the antibodies or antigen-binding fragments exemplified hereinare murine. In some embodiments, antibodies or antigen-binding fragmentsmay include a light chain CDR1 amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 18. In some embodiments, antibodiesor antigen-binding fragments may include a light chain CDR2 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 19. Insome embodiments, antibodies or antigen-binding fragments may include alight chain CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 20. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 22. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 23. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 24. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 18; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 19; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 20. Theantibodies or antigen-binding fragments may include a heavy chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 22; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 23; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 24. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 18; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 19; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 20, and also havea heavy chain having a CDR1 amino acid sequence substantially the sameas, or identical to, SEQ ID NO: 22; a CDR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 23; and a CDR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 24.

The described antibodies or antigen-binding fragments may include alight chain variable domain that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 21. In someembodiments, an isolated polynucleotide that includes a sequencesubstantially the same as, or identical to, SEQ ID NO: 53 may encodethis light chain variable domain amino acid sequence. The describedantibodies or antigen-binding fragments may include a heavy chainvariable domain that includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 25. In some embodiments, anisolated polynucleotide that includes a sequence substantially the sameas, or identical to, SEQ ID NO: 57 may encode this heavy chain variabledomain amino acid sequence. The described antibodies or antigen-bindingfragments may include a light and a heavy chain variable domains,wherein the light chain variable domain includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 21, and the heavychain variable domain includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 25.

In some embodiments, the antibodies are produced by antibody-producingcells deposited with the American Type Culture Collection (10801University Blvd., Manassas, Va. 20110-2209) on May 19, 2011 and havebeen assigned Accession No. PTA-11886. In some embodiments, theantibodies, or antigen-binding fragments thereof, have the bindingaffinity for FRα of the antibodies produced by the depositedantibody-producing cells. In some embodiments, the disclosed antibodies,or antigen-binding fragments thereof, comprise the light and heavy chainCDRs of the antibodies produced by the deposited antibody-producingcells. In some embodiments, the antibodies, or antigen-binding fragmentsthereof, comprise the light and heavy chain variable regions of theantibodies produced by the deposited antibody-producing cells.

Also disclosed are polynucleotides that encode antibodies orantigen-binding fragments that specifically bind to FRα. In someembodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR1 sequencesubstantially the same as, or identical to, SEQ ID NO: 18, for exampleSEQ ID NO: 50. In some embodiments, the isolated polynucleotides encodean antibody or antigen-binding fragment thereof having a light chainCDR2 substantially the same as, or identical to, SEQ ID NO: 19, forexample SEQ ID NO: 51. In some embodiments, the isolated polynucleotidesencode an antibody or antigen-binding fragment thereof having a lightchain CDR3 substantially the same as, or identical to, SEQ ID NO: 20,for example SEQ ID NO: 52. In some embodiments, the isolatedpolynucleotides encode an antibody or antigen-binding fragment thereofhaving a heavy chain CDR1 substantially the same as, or identical to,SEQ ID NO: 22, for example SEQ ID NO: 54. In some embodiments, theisolated polynucleotides encode an antibody or antigen-binding fragmentthereof having a heavy chain CDR2 substantially the same as, oridentical to, SEQ ID NO: 23, for example SEQ ID NO: 55. In someembodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR3 substantiallythe same as, or identical to, SEQ ID NO: 24, for example SEQ ID NO: 56.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a light chain with a CDR1 substantially the same as, oridentical to, SEQ ID NO: 18, for example SEQ ID NO: 50; a CDR2substantially the same as, or identical to, SEQ ID NO: 19, for exampleSEQ ID NO: 51; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 20, for example SEQ ID NO: 52. The polynucleotides may encodean antibody or antigen-binding fragment thereof having a heavy chainCDR1 substantially the same as, or identical to, SEQ ID NO: 22, forexample SEQ ID NO: 54; a CDR2 substantially the same as, or identicalto, SEQ ID NO: 23, for example SEQ ID NO: 55; and a CDR3 substantiallythe same as, or identical to, SEQ ID NO: 24, for example SEQ ID NO: 56.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a light chain CDR1 substantially the same as, oridentical to, SEQ ID NO: 18, for example SEQ ID NO: 50; a CDR2 encodedby a nucleotide sequence substantially the same as, or identical to, SEQID NO: 19, for example SEQ ID NO: 51; and a CDR3 encoded by a nucleotidesequence substantially the same as, or identical to, SEQ ID NO: 20, forexample SEQ ID NO: 52; and a heavy chain CDR1 substantially the same as,or identical to, SEQ ID NO: 22, for example SEQ ID NO: 54; a CDR2substantially the same as, or identical to, SEQ ID NO: 23, for exampleSEQ ID NO: 55; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 24, for example SEQ ID NO: 56. Antigen-binding arrangementsof CDRs may also be engineered using antibody-like proteins as CDRscaffolding. Such engineered antigen-binding proteins are within thescope of the disclosure.

Polynucleotides described herein may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 21, for example SEQ ID NO: 53. In someembodiments the described polynucleotides may encode antibodies orantigen-binding fragments that have a heavy chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 25, for example SEQ ID NO: 57. In someembodiments the described polynucleotides may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 21, for example SEQ ID NO: 53; and a heavychain variable domain segment that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 25, for exampleSEQ ID NO: 57. The polynucleotides capable of encoding the variabledomain segments provided herein may be included on the same, ordifferent, vectors to produce antibodies or antigen-binding fragments.

Described herein are isolated antibodies and antigen-binding fragmentsthat specifically bind to FRα. In some embodiments, the antibodies orantigen-binding fragments are murine IgG, or derivatives thereof. Whilethe antibodies or antigen-binding fragments may be human, humanized, orchimeric, the antibodies or antigen-binding fragments exemplified hereinare murine. In some embodiments, antibodies or antigen-binding fragmentsmay include a light chain CDR1 amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 26. In some embodiments, antibodiesor antigen-binding fragments may include a light chain CDR2 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 27. Insome embodiments, antibodies or antigen-binding fragments may include alight chain CDR3 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 28. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR1 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 30. Insome embodiments, antibodies or antigen-binding fragments may include aheavy chain CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 31. In some embodiments, antibodies orantigen-binding fragments may include a heavy chain CDR3 amino acidsequence substantially the same as, or identical to, SEQ ID NO: 32. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 26; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 27; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 28. Theantibodies or antigen-binding fragments may include a heavy chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 30; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 31; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 32. Theantibodies or antigen-binding fragments may include a light chain havinga CDR1 amino acid sequence substantially the same as, or identical to,SEQ ID NO: 26; a CDR2 amino acid sequence substantially the same as, oridentical to, SEQ ID NO: 27; and a CDR3 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 28, and also havea heavy chain having a CDR1 amino acid sequence substantially the sameas, or identical to, SEQ ID NO: 30; a CDR2 amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 31; and a CDR3amino acid sequence substantially the same as, or identical to, SEQ IDNO: 32.

The described antibodies or antigen-binding fragments may include alight chain variable domain that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 29. In someembodiments, an isolated polynucleotide that includes a sequencesubstantially the same as, or identical to, SEQ ID NO: 61 may encodethis light chain variable domain amino acid sequence. The describedantibodies or antigen-binding fragments may include a heavy chainvariable domain that includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 33. In some embodiments, anisolated polynucleotide that includes a sequence substantially the sameas, or identical to, SEQ ID NO: 65 may encode this heavy chain variabledomain amino acid sequence. The described antibodies or antigen-bindingfragments may include a light and a heavy chain variable domains,wherein the light chain variable domain includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 29, and the heavychain variable domain includes an amino acid sequence substantially thesame as, or identical to, SEQ ID NO: 33.

In some embodiments, the antibodies are produced by antibody-producingcells deposited with the American Type Culture Collection (10801University Blvd., Manassas, Va. 20110-2209) on May 19, 2011 and havebeen assigned Accession No. PTA-11885. In some embodiments, theantibodies, or antigen-binding fragments thereof, have the bindingaffinity for FRα of the antibodies produced by the depositedantibody-producing cells. In some embodiments, the disclosed antibodies,or antigen-binding fragments thereof, comprise the light and heavy chainCDRs of the antibodies produced by the deposited antibody-producingcells. In some embodiments, the antibodies, or antigen-binding fragmentsthereof, comprise the light and heavy chain variable regions of theantibodies produced by the deposited antibody-producing cells.

Also disclosed are polynucleotides that encode antibodies orantigen-binding fragments that specifically bind to FRα. In someembodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a light chain CDR1 sequencesubstantially the same as, or identical to, SEQ ID NO: 26, for exampleSEQ ID NO: 58. In some embodiments, the isolated polynucleotides encodean antibody or antigen-binding fragment thereof having a light chainCDR2 substantially the same as, or identical to, SEQ ID NO: 27, forexample SEQ ID NO: 59. In some embodiments, the isolated polynucleotidesencode an antibody or antigen-binding fragment thereof having a lightchain CDR3 substantially the same as, or identical to, SEQ ID NO: 28,for example SEQ ID NO: 60. In some embodiments, the isolatedpolynucleotides encode an antibody or antigen-binding fragment thereofhaving a heavy chain CDR1 substantially the same as, or identical to,SEQ ID NO: 30, for example SEQ ID NO: 62. In some embodiments, theisolated polynucleotides encode an antibody or antigen-binding fragmentthereof having a heavy chain CDR2 substantially the same as, oridentical to, SEQ ID NO: 31, for example SEQ ID NO: 63. In someembodiments, the isolated polynucleotides encode an antibody orantigen-binding fragment thereof having a heavy chain CDR3 substantiallythe same as, or identical to, SEQ ID NO: 32, for example SEQ ID NO: 64.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a light chain with a CDR1 substantially the same as, oridentical to, SEQ ID NO: 26, for example SEQ ID NO: 58; a CDR2substantially the same as, or identical to, SEQ ID NO: 27, for exampleSEQ ID NO: 59; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 28, for example SEQ ID NO: 60. The polynucleotides may encodean antibody or antigen-binding fragment thereof having a heavy chainCDR1 substantially the same as, or identical to, SEQ ID NO: 30, forexample SEQ ID NO: 62; a CDR2 substantially the same as, or identicalto, SEQ ID NO: 31, for example SEQ ID NO: 63; and a CDR3 substantiallythe same as, or identical to, SEQ ID NO: 32, for example SEQ ID NO: 64.The polynucleotides may encode an antibody or antigen-binding fragmentthereof having a light chain CDR1 substantially the same as, oridentical to, SEQ ID NO: 26, for example SEQ ID NO: 58; a CDR2 encodedby a nucleotide sequence substantially the same as, or identical to, SEQID NO: 27, for example SEQ ID NO: 59; and a CDR3 encoded by a nucleotidesequence substantially the same as, or identical to, SEQ ID NO: 28, forexample SEQ ID NO: 60; and a heavy chain CDR1 substantially the same as,or identical to, SEQ ID NO: 30, for example SEQ ID NO: 62; a CDR2substantially the same as, or identical to, SEQ ID NO: 31, for exampleSEQ ID NO: 63; and a CDR3 substantially the same as, or identical to,SEQ ID NO: 32, for example SEQ ID NO: 64. Antigen-binding arrangementsof CDRs may also be engineered using antibody-like proteins as CDRscaffolding. Such engineered antigen-binding proteins are within thescope of the disclosure.

Polynucleotides described herein may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 29, for example SEQ ID NO: 61. In someembodiments the described polynucleotides may encode antibodies orantigen-binding fragments that have a heavy chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 33, for example SEQ ID NO: 65. In someembodiments the described polynucleotides may encode antibodies orantigen-binding fragments that have a light chain variable domainsegment that includes an amino acid sequence substantially the same as,or identical to, SEQ ID NO: 29, for example SEQ ID NO: 61; and a heavychain variable domain segment that includes an amino acid sequencesubstantially the same as, or identical to, SEQ ID NO: 33, for exampleSEQ ID NO: 65. The polynucleotides capable of encoding the variabledomain segments provided herein may be included on the same, ordifferent, vectors to produce antibodies or antigen-binding fragments.

Polynucleotides encoding engineered antigen-binding proteins also arewithin the scope of the disclosure. In some embodiments, thepolynucleotides described (and the peptides they encode) include aleader sequence. Any leader sequence known in the art may be employed.The leader sequence may include, but is not limited to, a restrictionsite or a translation start site.

The antibodies or antigen-binding fragments described herein includevariants having single or multiple amino acid substitutions, deletions,or additions that retain the biological properties (e.g., bindingaffinity or immune effector activity) of the described antibodies orantigen-binding fragments. The skilled person may produce variantshaving single or multiple amino acid substitutions, deletions, oradditions. These variants may include: (a) variants in which one or moreamino acid residues are substituted with conservative or nonconservativeamino acids, (b) variants in which one or more amino acids are added toor deleted from the polypeptide, (c) variants in which one or more aminoacids include a substituent group, and (d) variants in which thepolypeptide is fused with another peptide or polypeptide such as afusion partner, a protein tag or other chemical moiety, that may conferuseful properties to the polypeptide, such as, for example, an epitopefor an antibody, a polyhistidine sequence, a biotin moiety and the like.Antibodies or antigen-binding fragments described herein may includevariants in which amino acid residues from one species are substitutedfor the corresponding residue in another species, either at theconserved or nonconserved positions. In other embodiments, amino acidresidues at nonconserved positions are substituted with conservative ornonconservative residues. The techniques for obtaining these variants,including genetic (suppressions, deletions, mutations, etc.), chemical,and enzymatic techniques, are known to the person having ordinary skillin the art.

The antibodies or antigen-binding fragments described herein may embodyseveral antibody isotypes, such as IgM, IgD, IgG, IgA and IgE. Antibodyor antigen-binding fragment thereof specificity is largely determined bythe amino acid sequence, and arrangement, of the CDRs. Therefore, theCDRs of one isotype may be transferred to another isotype withoutaltering antigen specificity. Alternatively, techniques have beenestablished to cause hybridomas to switch from producing one antibodyisotype to another (isotype switching) without altering antigenspecificity. Accordingly, such antibody isotypes are within the scope ofthe described antibodies or antigen-binding fragments

The antibodies or antigen-binding fragments described herein havebinding affinities (in M) for FRα that include a dissociation constant(K_(D)) of less than about 1×10⁻⁸ M. In one embodiment the antibody 9F3has an affinity for FRα of 7.15×10⁻¹⁰ M. In one embodiment the antibody19D4 has an affinity for FRα of 5.67×10⁻¹⁰ M. In one embodiment theantibody 24F12 has an affinity for FRα of 1.02×10⁻¹⁰ M. In oneembodiment the antibody 26B3 has an affinity for FRα of 2.73×10⁻¹¹ M. Inone embodiment the antibody 9F3 has an affinity for FRα of about6.5×10⁻¹⁰ M to about 8×10⁻¹⁰ M. In one embodiment the antibody 19D4 hasan affinity for FRα of about 5×10⁻¹⁰ M to about 6.5×10⁻¹⁰ M. In oneembodiment the antibody 24F12 has an affinity for FRα of about 0.5×10⁻¹⁰M to about 2×10⁻¹⁰ M. In one embodiment the antibody 26B3 has anaffinity for FRα of about 1×10⁻¹¹ M to about 3.5×10⁻¹¹ M.

Also provided are vectors comprising the polynucleotides describedherein. The vectors can be expression vectors. Recombinant expressionvectors containing a sequence encoding a polypeptide of interest arethus contemplated as within the scope of this disclosure. The expressionvector may contain one or more additional sequences such as but notlimited to regulatory sequences (e.g., promoter, enhancer), a selectionmarker, and a polyadenylation signal. Vectors for transforming a widevariety of host cells are well known and include, but are not limitedto, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterialartificial chromosomes (BACs), yeast artificial chromosomes (YACs), aswell as other bacterial, yeast and viral vectors.

Recombinant expression vectors within the scope of the descriptioninclude synthetic, genomic, or cDNA-derived nucleic acid fragments thatencode at least one recombinant protein which may be operably linked tosuitable regulatory elements. Such regulatory elements may include atranscriptional promoter, sequences encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. Expression vectors, especially mammalianexpression vectors, may also include one or more nontranscribed elementssuch as an origin of replication, a suitable promoter and enhancerlinked to the gene to be expressed, other 5′ or 3′ flankingnontranscribed sequences, 5′ or 3′ nontranslated sequences (such asnecessary ribosome binding sites), a polyadenylation site, splice donorand acceptor sites, or transcriptional termination sequences. An originof replication that confers the ability to replicate in a host may alsobe incorporated.

The transcriptional and translational control sequences in expressionvectors to be used in transforming vertebrate cells may be provided byviral sources. Exemplary vectors may be constructed as described byOkayama and Berg, 3 Mol. Cell. Biol. 280 (1983).

In some embodiments, the antibody- or antigen-binding fragment-codingsequence is placed under control of a powerful constitutive promoter,such as the promoters for the following genes: hypoxanthinephosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase,beta-actin, human myosin, human hemoglobin, human muscle creatine, andothers. In addition, many viral promoters function constitutively ineukaryotic cells and are suitable for use with the describedembodiments. Such viral promoters include without limitation,Cytomegalovirus (CMV) immediate early promoter, the early and latepromoters of SV40, the Mouse Mammary Tumor Virus (MMTV) promoter, thelong terminal repeats (LTRs) of Maloney leukemia virus, HumanImmunodeficiency Virus (HIV), Epstein Barr Virus (EBV), Rous SarcomaVirus (RSV), and other retroviruses, and the thymidine kinase promoterof Herpes Simplex Virus. In one embodiment, the antibody orantigen-binding fragment thereof coding sequence is placed under controlof an inducible promoter such as the metallothionein promoter,tetracycline-inducible promoter, doxycycline-inducible promoter,promoters that contain one or more interferon-stimulated responseelements (ISRE) such as protein kinase R 2′,5′-oligoadenylatesynthetases, Mx genes, ADAR1, and the like.

Vectors described herein may contain one or more Internal Ribosome EntrySite(s) (IRES). Inclusion of an IRES sequence into fusion vectors may bebeneficial for enhancing expression of some proteins. In someembodiments the vector system will include one or more polyadenylationsites (e.g., SV40), which may be upstream or downstream of any of theaforementioned nucleic acid sequences. Vector components may becontiguously linked, or arranged in a manner that provides optimalspacing for expressing the gene products (i.e., by the introduction of“spacer” nucleotides between the ORFs), or positioned in another way.Regulatory elements, such as the IRES motif, may also be arranged toprovide optimal spacing for expression.

The vectors may comprise selection markers, which are well known in theart. Selection markers include positive and negative selection markers,for example, antibiotic resistance genes (e.g., neomycin resistancegene, a hygromycin resistance gene, a kanamycin resistance gene, atetracycline resistance gene, a penicillin resistance gene), glutamatesythase genes, HSV-TK, HSV-TK derivatives for ganciclovir selection, orbacterial purine nucleoside phosphorylase gene for 6-methylpurineselection (Gadi et al., 7 Gene Ther. 1738-1743 (2000)). A nucleic acidsequence encoding a selection marker or the cloning site may be upstreamor downstream of a nucleic acid sequence encoding a polypeptide ofinterest or cloning site.

The vectors described herein may be used to transform various cells withthe genes encoding the described antibodies or antigen-bindingfragments. For example, the vectors may be used to generate antibody orantigen-binding fragment-producing cells. Thus, another aspect featureshost cells transformed with vectors comprising a nucleic acid sequenceencoding an antibody or antigen-binding fragment thereof thatspecifically binds FRα, such as the antibodies or antigen-bindingfragments described and exemplified herein.

Numerous techniques are known in the art for the introduction of foreigngenes into cells and may be used to construct the recombinant cells forpurposes of carrying out the described methods, in accordance with thevarious embodiments described and exemplified herein. The technique usedshould provide for the stable transfer of the heterologous gene sequenceto the host cell, such that the heterologous gene sequence is heritableand expressible by the cell progeny, and so that the necessarydevelopment and physiological functions of the recipient cells are notdisrupted. Techniques which may be used include but are not limited tochromosome transfer (e.g., cell fusion, chromosome mediated genetransfer, micro cell mediated gene transfer), physical methods (e.g.,transfection, spheroplast fusion, microinjection, electroporation,liposome carrier), viral vector transfer (e.g., recombinant DNA viruses,recombinant RNA viruses) and the like (described in Cline, 29 Pharmac.Ther. 69-92 (1985)). Calcium phosphate precipitation and polyethyleneglycol (PEG)-induced fusion of bacterial protoplasts with mammaliancells may also be used to transform cells.

Cells suitable for use in the expression of the antibodies orantigen-binding fragments described herein are preferably eukaryoticcells, more preferably cells of plant, rodent, or human origin, forexample but not limited to NSO, CHO, CHOK1, PERC.6® production cellline, Tk-ts13, BHK, HEK293 cells, COS-7, T98G, CV-1/EBNA, L cells, C127,3T3, HeLa, NS1, Sp2/0 myeloma cells, and BHK cell lines, among others.In addition, expression of antibodies may be accomplished usinghybridoma cells. Methods for producing hybridomas are well establishedin the art.

Cells transformed with expression vectors described herein may beselected or screened for recombinant expression of the antibodies orantigen-binding fragments described herein. Recombinant-positive cellsare expanded and screened for subclones exhibiting a desired phenotype,such as high level expression, enhanced growth properties, or theability to yield proteins with desired biochemical characteristics, forexample, due to protein modification or altered post-translationalmodifications. These phenotypes may be due to inherent properties of agiven subclone or to mutation. Mutations may be effected through the useof chemicals, UV-wavelength light, radiation, viruses, insertionalmutagens, inhibition of DNA mismatch repair, or a combination of suchmethods.

Provided herein are methods for detecting FRα in a sample by contactingthe sample with an antibody, or antigen-binding fragment thereof,described herein. As described herein, the sample may be derived fromurine, blood, serum, plasma, saliva, ascites, circulating cells,circulating tumor cells, cells that are not tissue associated (i.e.,free cells), tissues (e.g., surgically resected tumor tissue, biopsies,including fine needle aspiration), histological preparations, and thelike. In some embodiments the described methods include detecting FRα ina sample by contacting the sample with:

(a) an antibody, or antigen-binding fragment thereof, that binds thesame epitope as any one of antibody 9F3, antibody 19D4, antibody 24F12,or antibody 26B3;

(b) any one of antibody 9F3, antibody 19D4, antibody 24F12, or antibody26B3, or an antigen-binding fragment thereof;

(c) an antibody, or antigen-binding fragment thereof, that comprisesheavy chain CDR1, CDR2, and CDR3 amino acid sequences and light chainCDR1, CDR2, and CDR3 amino acid sequences of any one of antibody 9F3,antibody 19D4, antibody 24F12, or antibody 26B3, as described in Table1;

(d) an antibody, or antigen-binding fragment thereof, that comprises theheavy chain variable domain segment and light chain variable domainsegment of any one of antibody 9F3, antibody 19D4, antibody 24F12, orantibody 26B3, as described in Table 1; or

(e) an antibody having the amino acid sequence of antibody produced byany one of the cell lines deposited with the ATCC having accessionnumber PTA-11887, PTA-11884, PTA-11886, or PTA-11885, or an antigenbinding fragment thereof.

In some embodiments the sample may be contacted with more than one ofthe antibodies, or antigen-binding fragments described herein. Forexample, a sample may be contacted with a first antibody, orantigen-binding fragment thereof, and then contacted with a secondantibody, or antigen-binding fragment thereof, wherein the firstantibody or antigen-binding fragment and the second antibody orantigen-binding fragment are not the same antibody or antigen-bindingfragment. In some embodiments, the first antibody, or antigen-bindingfragment thereof, may be affixed to a surface, such as a multiwellplate, chip, or similar substrate prior to contacting the sample. Inother embodiments the first antibody, or antigen-binding fragmentthereof, may not be affixed, or attached, to anything at all prior tocontacting the sample.

Various combinations of the antibodies, or antigen-binding fragmentsthereof, may be used to detect FRα in a sample. In one embodiment thesample may be first contacted with an antibody, or antigen-bindingfragment thereof, that comprises heavy chain CDR1, CDR2, and CDR3 aminoacid sequences and light chain CDR1, CDR2, and CDR3 amino acid sequencesof antibody 9F3 (as provided in Table 1), and then separately contactedwith a second antibody, or antigen-binding fragment thereof, thatcomprises heavy chain CDR1, CDR2, and CDR3 amino acid sequences andlight chain CDR1, CDR2, and CDR3 amino acid sequences of antibody 19D4(as provided in Table 1). In one embodiment the sample may be firstcontacted with an antibody, or antigen-binding fragment thereof, thatcomprises heavy chain CDR1, CDR2, and CDR3 amino acid sequences andlight chain CDR1, CDR2, and CDR3 amino acid sequences of antibody 9F3(as provided in Table 1), and then separately contacted with a secondantibody, or antigen-binding fragment thereof, that comprises heavychain CDR1, CDR2, and CDR3 amino acid sequences and light chain CDR1,CDR2, and CDR3 amino acid sequences of antibody 24F12 (as provided inTable 1). In one embodiment the sample may be first contacted with anantibody, or antigen-binding fragment thereof, that comprises heavychain CDR1, CDR2, and CDR3 amino acid sequences and light chain CDR1,CDR2, and CDR3 amino acid sequences of antibody 9F3 (as provided inTable 1), and then separately contacted with a second antibody, orantigen-binding fragment thereof, that comprises heavy chain CDR1, CDR2,and CDR3 amino acid sequences and light chain CDR1, CDR2, and CDR3 aminoacid sequences of antibody 26B3 (as provided in Table 1). In oneembodiment the sample may be first contacted with an antibody, orantigen-binding fragment thereof, that comprises heavy chain CDR1, CDR2,and CDR3 amino acid sequences and light chain CDR1, CDR2, and CDR3 aminoacid sequences of antibody 19D4 (as provided in Table 1), and thenseparately contacted with a second antibody, or antigen-binding fragmentthereof, that comprises heavy chain CDR1, CDR2, and CDR3 amino acidsequences and light chain CDR1, CDR2, and CDR3 amino acid sequences ofantibody 9F3 (as provided in Table 1). In one embodiment the sample maybe first contacted with an antibody, or antigen-binding fragmentthereof, that comprises heavy chain CDR1, CDR2, and CDR3 amino acidsequences and light chain CDR1, CDR2, and CDR3 amino acid sequences ofantibody 19D4 (as provided in Table 1), and then separately contactedwith a second antibody, or antigen-binding fragment thereof, thatcomprises heavy chain CDR1, CDR2, and CDR3 amino acid sequences andlight chain CDR1, CDR2, and CDR3 amino acid sequences of antibody 24F12(as provided in Table 1). In one embodiment the sample may be firstcontacted with an antibody, or antigen-binding fragment thereof, thatcomprises heavy chain CDR1, CDR2, and CDR3 amino acid sequences andlight chain CDR1, CDR2, and CDR3 amino acid sequences of antibody 19D4(as provided in Table 1), and then separately contacted with a secondantibody, or antigen-binding fragment thereof, that comprises heavychain CDR1, CDR2, and CDR3 amino acid sequences and light chain CDR1,CDR2, and CDR3 amino acid sequences of antibody 26B3 (as provided inTable 1). In one embodiment the sample may be first contacted with anantibody, or antigen-binding fragment thereof, that comprises heavychain CDR1, CDR2, and CDR3 amino acid sequences and light chain CDR1,CDR2, and CDR3 amino acid sequences of antibody 24F12 (as provided inTable 1), and then separately contacted with a second antibody, orantigen-binding fragment thereof, that comprises heavy chain CDR1, CDR2,and CDR3 amino acid sequences and light chain CDR1, CDR2, and CDR3 aminoacid sequences of antibody 9F3 (as provided in Table 1). In oneembodiment the sample may be first contacted with an antibody, orantigen-binding fragment thereof, that comprises heavy chain CDR1, CDR2,and CDR3 amino acid sequences and light chain CDR1, CDR2, and CDR3 aminoacid sequences of antibody 24F12 (as provided in Table 1), and thenseparately contacted with a second antibody, or antigen-binding fragmentthereof, that comprises heavy chain CDR1, CDR2, and CDR3 amino acidsequences and light chain CDR1, CDR2, and CDR3 amino acid sequences ofantibody 19D4 (as provided in Table 1). In one embodiment the sample maybe first contacted with an antibody, or antigen-binding fragmentthereof, that comprises heavy chain CDR1, CDR2, and CDR3 amino acidsequences and light chain CDR1, CDR2, and CDR3 amino acid sequences ofantibody 24F12 (as provided in Table 1), and then separately contactedwith a second antibody, or antigen-binding fragment thereof, thatcomprises heavy chain CDR1, CDR2, and CDR3 amino acid sequences andlight chain CDR1, CDR2, and CDR3 amino acid sequences of antibody 26B3(as provided in Table 1). In one embodiment the sample may be firstcontacted with an antibody, or antigen-binding fragment thereof, thatcomprises heavy chain CDR1, CDR2, and CDR3 amino acid sequences andlight chain CDR1, CDR2, and CDR3 amino acid sequences of antibody 26B3(as provided in Table 1), and then separately contacted with a secondantibody, or antigen-binding fragment thereof, that comprises heavychain CDR1, CDR2, and CDR3 amino acid sequences and light chain CDR1,CDR2, and CDR3 amino acid sequences of antibody 9F3 (as provided inTable 1). In one embodiment the sample may be first contacted with anantibody, or antigen-binding fragment thereof, that comprises heavychain CDR1, CDR2, and CDR3 amino acid sequences and light chain CDR1,CDR2, and CDR3 amino acid sequences of antibody 26B3 (as provided inTable 1), and then separately contacted with a second antibody, orantigen-binding fragment thereof, that comprises heavy chain CDR1, CDR2,and CDR3 amino acid sequences and light chain CDR1, CDR2, and CDR3 aminoacid sequences of antibody 24F12 (as provided in Table 1). In oneembodiment the sample may be first contacted with an antibody, orantigen-binding fragment thereof, that comprises heavy chain CDR1, CDR2,and CDR3 amino acid sequences and light chain CDR1, CDR2, and CDR3 aminoacid sequences of antibody 26B3 (as provided in Table 1), and thenseparately contacted with a second antibody, or antigen-binding fragmentthereof, that comprises heavy chain CDR1, CDR2, and CDR3 amino acidsequences and light chain CDR1, CDR2, and CDR3 amino acid sequences ofantibody 19D4 (as provided in Table 1). In one embodiment the sample maybe first contacted with an antibody, or antigen-binding fragmentthereof, that comprises the heavy chain variable domain segment andlight chain variable domain segment amino acid sequences of antibody 9F3(as provided in Table 1), and then separately contacted with a secondantibody, or antigen-binding fragment thereof, that comprises the heavychain variable domain segment and light chain variable domain segmentamino acid sequences of antibody 19D4 (as provided in Table 1). In oneembodiment the sample may be first contacted with an antibody, orantigen-binding fragment thereof, that comprises the heavy chainvariable domain segment and light chain variable domain segment aminoacid sequences of antibody 9F3 (as provided in Table 1), and thenseparately contacted with a second antibody, or antigen-binding fragmentthereof, that comprises the heavy chain variable domain segment andlight chain variable domain segment amino acid sequences of antibody24F12 (as provided in Table 1). In one embodiment the sample may befirst contacted with an antibody, or antigen-binding fragment thereof,that comprises the heavy chain variable domain segment and light chainvariable domain segment amino acid sequences of antibody 9F3 (asprovided in Table 1), and then separately contacted with a secondantibody, or antigen-binding fragment thereof, that comprises the heavychain variable domain segment and light chain variable domain segmentamino acid sequences of antibody 26B3 (as provided in Table 1). In oneembodiment the sample may be first contacted with an antibody, orantigen-binding fragment thereof, that comprises the heavy chainvariable domain segment and light chain variable domain segment aminoacid sequences of antibody 19D4 (as provided in Table 1), and thenseparately contacted with a second antibody, or antigen-binding fragmentthereof, that comprises the heavy chain variable domain segment andlight chain variable domain segment amino acid sequences of antibody 9F3(as provided in Table 1). In one embodiment the sample may be firstcontacted with an antibody, or antigen-binding fragment thereof, thatcomprises the heavy chain variable domain segment and light chainvariable domain segment amino acid sequences of antibody 19D4 (asprovided in Table 1), and then separately contacted with a secondantibody, or antigen-binding fragment thereof, that comprises the heavychain variable domain segment and light chain variable domain segmentamino acid sequences of antibody 24F12 (as provided in Table 1). In oneembodiment the sample may be first contacted with an antibody, orantigen-binding fragment thereof, that comprises the heavy chainvariable domain segment and light chain variable domain segment aminoacid sequences of antibody 19D4 (as provided in Table 1), and thenseparately contacted with a second antibody, or antigen-binding fragmentthereof, that comprises the heavy chain variable domain segment andlight chain variable domain segment amino acid sequences of antibody26B3 (as provided in Table 1). In one embodiment the sample may be firstcontacted with an antibody, or antigen-binding fragment thereof, thatcomprises the heavy chain variable domain segment and light chainvariable domain segment amino acid sequences of antibody 24F12 (asprovided in Table 1), and then separately contacted with a secondantibody, or antigen-binding fragment thereof, that comprises the heavychain variable domain segment and light chain variable domain segmentamino acid sequences of antibody 9F3 (as provided in Table 1). In oneembodiment the sample may be first contacted with an antibody, orantigen-binding fragment thereof, that comprises the heavy chainvariable domain segment and light chain variable domain segment aminoacid sequences of antibody 24F12 (as provided in Table 1), and thenseparately contacted with a second antibody, or antigen-binding fragmentthereof, that comprises the heavy chain variable domain segment andlight chain variable domain segment amino acid sequences of antibody19D4 (as provided in Table 1). In one embodiment the sample may be firstcontacted with an antibody, or antigen-binding fragment thereof, thatcomprises the heavy chain variable domain segment and light chainvariable domain segment amino acid sequences of antibody 24F12 (asprovided in Table 1), and then separately contacted with a secondantibody, or antigen-binding fragment thereof, that comprises the heavychain variable domain segment and light chain variable domain segmentamino acid sequences of antibody 26B3 (as provided in Table 1). In oneembodiment the sample may be first contacted with an antibody, orantigen-binding fragment thereof, that comprises the heavy chainvariable domain segment and light chain variable domain segment aminoacid sequences of antibody 26B3 (as provided in Table 1), and thenseparately contacted with a second antibody, or antigen-binding fragmentthereof, that comprises the heavy chain variable domain segment andlight chain variable domain segment amino acid sequences of antibody 9F3(as provided in Table 1). In one embodiment the sample may be firstcontacted with an antibody, or antigen-binding fragment thereof, thatcomprises the heavy chain variable domain segment and light chainvariable domain segment amino acid sequences of antibody 26B3 (asprovided in Table 1), and then separately contacted with a secondantibody, or antigen-binding fragment thereof, that comprises the heavychain variable domain segment and light chain variable domain segmentamino acid sequences of antibody 24F12 (as provided in Table 1). In oneembodiment the sample may be first contacted with an antibody, orantigen-binding fragment thereof, that comprises the heavy chainvariable domain segment and light chain variable domain segment aminoacid sequences of antibody 26B3 (as provided in Table 1), and thenseparately contacted with a second antibody, or antigen-binding fragmentthereof, that comprises the heavy chain variable domain segment andlight chain variable domain segment amino acid sequences of antibody19D4 (as provided in Table 1). In one embodiment the sample may be firstcontacted with the antibody produced by the cell line having ATCCaccession number PTA-11887, or an antigen-binding fragment thereof, andthen separately contacted with a second antibody produced by the cellline having ATCC accession number PTA-11884, or an antigen-bindingfragment thereof. In one embodiment the sample may be first contactedwith the antibody produced by the cell line having ATCC accession numberPTA-11887, or an antigen-binding fragment thereof, and then separatelycontacted with a second antibody produced by the cell line having ATCCaccession number PTA-11885, or an antigen-binding fragment thereof. Inone embodiment the sample may be first contacted with the antibodyproduced by the cell line having ATCC accession number PTA-11887, or anantigen-binding fragment thereof, and then separately contacted with asecond antibody produced by the cell line having ATCC accession numberPTA-11886, or an antigen-binding fragment thereof. In one embodiment thesample may be first contacted with the antibody produced by the cellline having ATCC accession number PTA-11884, or an antigen-bindingfragment thereof, and then separately contacted with a second antibodyproduced by the cell line having ATCC accession number PTA-11887, or anantigen-binding fragment thereof. In one embodiment the sample may befirst contacted with the antibody produced by the cell line having ATCCaccession number PTA-11884, or an antigen-binding fragment thereof, andthen separately contacted with a second antibody produced by the cellline having ATCC accession number PTA-11885, or an antigen-bindingfragment thereof. In one embodiment the sample may be first contactedwith the antibody produced by the cell line having ATCC accession numberPTA-11884, or an antigen-binding fragment thereof, and then separatelycontacted with a second antibody produced by the cell line having ATCCaccession number PTA-11886, or an antigen-binding fragment thereof. Inone embodiment the sample may be first contacted with the antibodyproduced by the cell line having ATCC accession number PTA-11885, or anantigen-binding fragment thereof, and then separately contacted with asecond antibody produced by the cell line having ATCC accession numberPTA-11884, or an antigen-binding fragment thereof. In one embodiment thesample may be first contacted with the antibody produced by the cellline having ATCC accession number PTA-11885, or an antigen-bindingfragment thereof, and then separately contacted with a second antibodyproduced by the cell line having ATCC accession number PTA-11887, or anantigen-binding fragment thereof. In one embodiment the sample may befirst contacted with the antibody produced by the cell line having ATCCaccession number PTA-11885, or an antigen-binding fragment thereof, andthen separately contacted with a second antibody produced by the cellline having ATCC accession number PTA-11886, or an antigen-bindingfragment thereof. In one embodiment the sample may be first contactedwith the antibody produced by the cell line having ATCC accession numberPTA-11886, or an antigen-binding fragment thereof, and then separatelycontacted with a second antibody produced by the cell line having ATCCaccession number PTA-11884, or an antigen-binding fragment thereof. Inone embodiment the sample may be first contacted with the antibodyproduced by the cell line having ATCC accession number PTA-11886, or anantigen-binding fragment thereof, and then separately contacted with asecond antibody produced by the cell line having ATCC accession numberPTA-11885, or an antigen-binding fragment thereof. In one embodiment thesample may be first contacted with the antibody produced by the cellline having ATCC accession number PTA-11886, or an antigen-bindingfragment thereof, and then separately contacted with a second antibodyproduced by the cell line having ATCC accession number PTA-11887, or anantigen-binding fragment thereof. In one embodiment the sample may befirst contacted with an antibody capable of binding to the same epitopeas the antibody produced by the cell line having ATCC accession numberPTA-11887, or an antigen-binding fragment thereof, and then separatelycontacted with a second antibody capable of binding to the same epitopeas antibody produced by the cell line having ATCC accession numberPTA-11884, or an antigen-binding fragment thereof. In one embodiment thesample may be first contacted with an antibody capable of binding to thesame epitope as the antibody produced by the cell line having ATCCaccession number PTA-11887, or an antigen-binding fragment thereof, andthen separately contacted with a second antibody capable of binding tothe same epitope as antibody produced by the cell line having ATCCaccession number PTA-11885, or an antigen-binding fragment thereof. Inone embodiment the sample may be first contacted with an antibodycapable of binding to the same epitope as the antibody produced by thecell line having ATCC accession number PTA-11887, or an antigen-bindingfragment thereof, and then separately contacted with a second antibodycapable of binding to the same epitope as antibody produced by the cellline having ATCC accession number PTA-11886, or an antigen-bindingfragment thereof. In one embodiment the sample may be first contactedwith an antibody capable of binding to the same epitope as the antibodyproduced by the cell line having ATCC accession number PTA-11884, or anantigen-binding fragment thereof, and then separately contacted with asecond antibody capable of binding to the same epitope as antibodyproduced by the cell line having ATCC accession number PTA-11887, or anantigen-binding fragment thereof. In one embodiment the sample may befirst contacted with an antibody capable of binding to the same epitopeas the antibody produced by the cell line having ATCC accession numberPTA-11884, or an antigen-binding fragment thereof, and then separatelycontacted with a second antibody capable of binding to the same epitopeas antibody produced by the cell line having ATCC accession numberPTA-11885, or an antigen-binding fragment thereof. In one embodiment thesample may be first contacted with an antibody capable of binding to thesame epitope as the antibody produced by the cell line having ATCCaccession number PTA-11884, or an antigen-binding fragment thereof, andthen separately contacted with a second antibody capable of binding tothe same epitope as antibody produced by the cell line having ATCCaccession number PTA-11886, or an antigen-binding fragment thereof. Inone embodiment the sample may be first contacted with an antibodycapable of binding to the same epitope as the antibody produced by thecell line having ATCC accession number PTA-11885, or an antigen-bindingfragment thereof, and then separately contacted with a second antibodycapable of binding to the same epitope as antibody produced by the cellline having ATCC accession number PTA-11884, or an antigen-bindingfragment thereof. In one embodiment the sample may be first contactedwith an antibody capable of binding to the same epitope as the antibodyproduced by the cell line having ATCC accession number PTA-11885, or anantigen-binding fragment thereof, and then separately contacted with asecond antibody capable of binding to the same epitope as antibodyproduced by the cell line having ATCC accession number PTA-11887, or anantigen-binding fragment thereof. In one embodiment the sample may befirst contacted with an antibody capable of binding to the same epitopeas the antibody produced by the cell line having ATCC accession numberPTA-11885, or an antigen-binding fragment thereof, and then separatelycontacted with a second antibody capable of binding to the same epitopeas antibody produced by the cell line having ATCC accession numberPTA-11886, or an antigen-binding fragment thereof. In one embodiment thesample may be first contacted with an antibody capable of binding to thesame epitope as the antibody produced by the cell line having ATCCaccession number PTA-11886, or an antigen-binding fragment thereof, andthen separately contacted with a second antibody capable of binding tothe same epitope as antibody produced by the cell line having ATCCaccession number PTA-11884, or an antigen-binding fragment thereof. Inone embodiment the sample may be first contacted with an antibodycapable of binding to the same epitope as the antibody produced by thecell line having ATCC accession number PTA-11886, or an antigen-bindingfragment thereof, and then separately contacted with a second antibodycapable of binding to the same epitope as antibody produced by the cellline having ATCC accession number PTA-11885, or an antigen-bindingfragment thereof. In one embodiment the sample may be first contactedwith an antibody capable of binding to the same epitope as the antibodyproduced by the cell line having ATCC accession number PTA-11886, or anantigen-binding fragment thereof, and then separately contacted with asecond antibody capable of binding to the same epitope as antibodyproduced by the cell line having ATCC accession number PTA-11887, or anantigen-binding fragment thereof.

The described antibodies and antigen-binding fragments may be detectablylabeled. In some embodiments labeled antibodies and antigen-bindingfragments may facilitate the detection FRα via the methods describedherein. Many such labels are readily known to those skilled in the art.For example, suitable labels include, but should not be consideredlimited to, radiolabels, fluorescent labels (such as DYLIGHT® 649fluorescent dye), epitope tags, biotin, chromophore labels, ECL labels,or enzymes. More specifically, the described labels include ruthenium,¹¹¹In-DOTA, ¹¹¹In-diethylenetriaminepentaacetic acid (DTPA), horseradishperoxidase, alkaline phosphatase and beta-galactosidase, poly-histidine(HIS tag), acridine dyes, cyanine dyes, fluorone dyes, oxazin dyes,phenanthridine dyes, rhodamine dyes, ALEXAFLUOR® dyes, and the like.

The described antibodies and antigen-binding fragments may be used in avariety of assays to detect FRα in a sample. Some suitable assaysinclude, but should not be considered limited to, western blot analysis,radioimmunoassay, immunofluorimetry, immunoprecipitation, equilibriumdialysis, immunodiffusion, electrochemiluminescence (ECL) immunoassay,immunohistochemistry, fluorescence-activated cell sorting (FACS) orELISA assay.

In some embodiments described herein detection of FRα-expressing cancercells in a subject may be used to determine that the subject may betreated with a therapeutic agent directed against FRα. In someembodiments the therapeutic agent directed against FRα may be anantibody, such as Farletuzumab.

Methods for Diagnosing Cancer

Provided herein are methods for diagnosing ovarian, breast, thyroid,colorectal, endometrial, fallopian tube, or lung cancer of epithelialorigin in a subject. In some embodiments, as described above, detectingFRα in a sample, such as a histological sample, a fine needle aspiratesample, resected tumor tissue, circulating cells, circulating tumorcells, and the like, provides the ability to diagnose cancer in thesubject from whom the sample was obtained. In some embodiments, it mayalready be known that the subject from whom the sample was obtained hascancer, but the type of cancer afflicting the subject may not yet havebeen diagnosed or a preliminary diagnosis may be unclear, thus detectingFRα in a sample obtained from the subject can allow for, or clarify,diagnosis of the cancer.

In some embodiments the described methods involve assessing whether asubject is afflicted with FRα-expressing cancer by determining theamount of FRα that is present in a sample derived from the subject; andcomparing the observed amount of FRα with the amount of FRα in a controlsample, wherein a difference between the amount of FRα in the samplederived from the subject and the amount of FRα in the control sample isan indication that the subject is afflicted with an FRα-expressingcancer. In some embodiments the amount of FRα in the sample derived fromthe subject is assessed by contacting the sample with an antibody thatbinds FRα, such as the antibodies described herein. Similar methods maybe used to determine if a subject is afflicted with cancer that is notassociated with increased FRα production. The sample assessed for thepresence of FRα may be derived from urine, blood, serum, plasma, saliva,ascites, circulating cells, circulating tumor cells, cells that are nottissue associated (i.e., free cells), tissues (e.g., surgically resectedtumor tissue, biopsies, including fine needle aspiration), histologicalpreparations, and the like. In some embodiments the subject is a human.

In some embodiments the method of diagnosing an FRα-expressing cancerwill involve: contacting a biological sample of a subject with anFRα-specific antibody, or antigen-binding fragment thereof (such asthose derivable from the antibodies and fragments provided in Table 1),quantifying the amount of FRα present in the sample that is bound by theantibody or antigen-binding fragment thereof, comparing the amount ofFRα present in the sample to a known standard; and determining whetherthe subject's FRα levels fall within the levels of FRα associated withcancer. In an additional embodiment, the diagnostic method can befollowed with an additional step of administering or prescribing acancer-specific treatment. In some embodiments the cancer-specifictreatment may be directed against FRα-expressing cancers, such asFarletuzumab.

In some embodiments the described methods involve assessing whether asubject is afflicted with FRα-expressing cancer by determining theamount of FRα associated with a cell or tissue that is present in asample derived from the subject; and comparing the observed amount ofFRα with the amount of FRα in a control sample, wherein a differencebetween the amount of FRα in the sample derived from the subject and theamount of FRα in the control sample is an indication that the subject isafflicted with an FRα-expressing cancer.

In some embodiments the control sample may be derived from a subjectthat is not afflicted with FRα-expressing cancer. In some embodimentsthe control sample may be derived from a subject that is afflicted withFRα-expressing cancer. In some embodiments where the control sample isderived from a subject that is not afflicted with FRα-expressing cancer,an observed increase in the amount of FRα present in the sample,relative to that observed for the control sample, is an indication thatthe subject being assessed is afflicted with FRα-expressing cancer. Insome embodiments where the control sample is derived from a subject thatis not afflicted with FRα-expressing cancer, an observed decrease orsimilarity in the amount of FRα present in the test sample, relative tothat observed for the control sample, is an indication that the subjectbeing assessed is not afflicted with FRα-expressing cancer. In someembodiments where the control sample is derived from a subject that isafflicted with FRα-expressing cancer, an observed similarity in theamount of FRα present in the test sample, relative to that observed forthe control sample, is an indication that the subject being assessed isafflicted with FRα-expressing cancer. In some embodiments where thecontrol sample is derived from a subject that is afflicted withFRα-expressing cancer, an observed decrease in the amount of FRα presentin the test sample, relative to that observed for the control sample, isan indication that the subject being assessed is not afflicted withFRα-expressing cancer.

In some embodiments the amount of FRα in the sample derived from thesubject is assessed by contacting the sample with an antibody that bindsFRα, such as the antibodies described herein. The sample assessed forthe presence of FRα may be circulating cells, circulating tumor cells,cells that are not tissue associated (i.e., free cells), tissues (e.g.,surgically resected tumor tissue, biopsies, including fine needleaspiration), histological preparations, and the like.

In some embodiments the described methods involve assessing whether asubject is afflicted with FRα-expressing cancer by determining theamount of FRα that is not associated with a cell or tissue that ispresent in a sample derived from the subject; and comparing the observedamount of FRα with the amount of FRα in a control sample, wherein adifference between the amount of FRα in the sample derived from thesubject and the amount of FRα in the control sample is an indicationthat the subject is afflicted with an FRα-expressing cancer. In someembodiments the amount of FRα in the sample derived from the subject isassessed by contacting the sample with an antibody that binds FRα, suchas the antibodies described herein. The sample assessed for the presenceof FRα may be urine, blood, serum, plasma, saliva, ascites, histologicalpreparations, and the like.

In various embodiments of the described methods, the cancer may beFRα-expressing cancer. In a particular embodiment, the FRα-expressingcancer is ovarian cancer. In some embodiments the FRα-expressing canceris endometrial cancer. In some embodiments the FRα-expressing cancer iscolorectal cancer. In some embodiments the FRα-expressing cancer isbreast cancer. In some embodiments the FRα-expressing cancer is thyroidcancer. In some embodiments the FRα-expressing cancer is fallopian tubecancer. In another embodiment, the FRα-expressing cancer is non-smallcell lung cancer, such as an adenocarcinoma. Alternatively, thedescribed methods may be used to diagnose cancer that does not expressFRα, such as squamous cell carcinoma.

In various aspects, the amount of FRα is determined by contacting thesample with an antibody, or antigen-binding fragment thereof, that bindsFRα. In some embodiments, the sample may be contacted by more than onetype of antibody, or antigen-binding fragment thereof, that binds FRα.In some embodiments, the sample may be contacted by a first antibody, orantigen-binding fragment thereof, that binds FRα and then contacted by asecond antibody, or antigen-binding fragment thereof, that binds FRα.Antibodies such as those described herein may be used in this capacity.For example, the antibody is selected from the group consisting of:

(a) an antibody, or antigen-binding fragment thereof, that binds thesame epitope as any one of antibody 9F3, antibody 19D4, antibody 24F12,or antibody 26B3;

(b) any one of antibody 9F3, antibody 19D4, antibody 24F12, or antibody26B3, or an antigen-binding fragment thereof;

(c) an antibody, or antigen-binding fragment thereof, that comprisesheavy chain CDR1, CDR2, and CDR3 amino acid sequences and light chainCDR1, CDR2, and CDR3 amino acid sequences of any one of antibody 9F3,antibody 19D4, antibody 24F12, or antibody 26B3, as described in Table1;

(d) an antibody, or antigen-binding fragment thereof, that comprises theheavy chain variable domain segment and light chain variable domainsegment of any one of antibody 9F3, antibody 19D4, antibody 24F12, orantibody 26B3, as described in Table 1; or

(e) an antibody having the amino acid sequence of antibody produced byany one of the cell lines deposited with the ATCC having accessionnumber PTA-11887, PTA-11884, PTA-11886, or PTA-11885, or an antigenbinding fragment thereof.

Various combinations of the antibodies and antigen-binding fragmentsdescribed in (a)-(e), as detailed above in the general sectiondescribing methods of detection, can be used to provide a “first” and“second” antibody or antigen-binding fragment to carry out the describeddiagnostic methods.

In certain embodiments, the amount of FRα is determined by western blotanalysis, radioimmunoassay, immunofluorimetry, immunoprecipitation,equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL)immunoassay, immunohistochemistry, fluorescence-activated cell sorting(FACS) or ELISA assay.

In various embodiments of the described diagnostic methods a controlsample is used. The control sample may be a positive or negative assaycontrol that ensures the assay used is working properly; for example, anassay control of this nature might be commonly used forimmunohistochemistry assays. Alternatively, the control sample may be astandardized control amount of FRα in a healthy subject. In someembodiments, the observed FRα levels of the tested subject may becompared with FRα levels observed in samples from control subjects knownto have FRα-expressing cancer. In some embodiments, the controlsubject's FRα-expressing cancer is ovarian cancer, endometrial cancer,colorectal cancer, breast cancer, thyroid cancer, fallopian tube cancer,or lung cancer, such as adenocarcinoma. In some embodiments, the controlsubject is known to have early stage FRα-expressing cancer, such asstage I ovarian cancer, endometrial cancer, colorectal cancer, breastcancer, thyroid cancer, fallopian tube cancer, or lung cancer (e.g.,adenocarcinoma). In some embodiments, the control subject is known tohave intermediate stage FRα-expressing cancer, such as stage II ovariancancer, endometrial cancer, colorectal cancer, breast cancer, thyroidcancer, fallopian tube cancer, or lung cancer (e.g., adenocarcinoma). Insome embodiments, the control subject is known to have late stageFRα-expressing cancer, such as stage III or stage IV ovarian cancer,endometrial cancer, colorectal cancer, breast cancer, thyroid cancer,fallopian tube cancer, or lung cancer (e.g., adenocarcinoma).

The diagnostic methods provided herein also provide a basis upon whichit may be possible to predict whether a subject has a relatively higheror lower likelihood of surviving 5 years following diagnosis. In someembodiments, the described method may be used to predict a favorableoutcome for a subject having adenocarcinoma, wherein a favorable outcomeis defined as having an increased 5-year survival rate. As data providedherein indicate, subjects determined to have stage I or stage IIadenocarcinoma that does not express FRα are about 2 times more likelyto die within five years than subjects determined to have stage I orstage II adenocarcinoma that does express FRα. Accordingly, thediagnostic methods described herein may be combined with this knowledgeto allow for a method of predicting 5-year survivorship likelihood forsubjects determined to have cancer. In some embodiments the method isused to predict the 5-year survivorship likelihood for subjectsdetermined to have adenocarcinoma.

In some embodiments the described prognostic method will involve:contacting a biological sample of a subject with an FRα-specificantibody, or antigen-binding fragment thereof (such as those derivablefrom the antibodies and fragments provided in Table 1), quantifying theamount of FRα present in the sample that is bound by the antibody orantigen-binding fragment thereof, comparing the amount of FRα present inthe sample to a known standard; and determining whether the subject'sFRα levels indicate the presence of a FRα expressing cancer, therebyallowing for a prediction to be made as to the likelihood the subjectwill survive five years after being diagnosed with cancer. In someembodiments the subject is known to have or determined to haveadenocarcinoma. In some embodiments the subject is a human.

Methods for Monitoring Cancer

Provided herein are methods for monitoring cancer of epithelial originin a subject. In some embodiments the described methods involveassessing whether FRα-expressing cancer is progressing, regressing, orremaining stable by determining the amount of FRα that is present in atest sample derived from the subject; and comparing the observed amountof FRα with the amount of FRα in a sample obtained from the subject atan earlier point in time, wherein a difference between the amount of FRαin the test sample and the earlier sample provides an indication ofwhether the cancer is progressing, regressing, or remaining stable. Inthis regard, a test sample with an increased amount of FRα, relative tothe amount observed for the earlier sample, may indicate progression ofan FRα-expressing cancer. Conversely, a test sample with a decreasedamount of FRα, relative to the amount observed for the earlier sample,may indicate regression of an FRα-expressing cancer. Accordingly, a testsample with an insignificant difference in the amount of FRα, relativeto the amount observed for the earlier sample, may indicate a state ofstable disease for an FRα-expressing cancer. In some embodiments theamount of FRα in a sample derived from the subject is assessed bycontacting the sample with an antibody that binds FRα, such as theantibodies described herein. The sample assessed for the presence of FRαmay be derived from urine, blood, serum, plasma, saliva, ascites,circulating cells, circulating tumor cells, cells that are not tissueassociated (i.e., free cells), tissues (e.g., surgically resected tumortissue, biopsies, including fine needle aspiration), histologicalpreparations, and the like. In some embodiments the subject is a human.

In some embodiments the method of monitoring an FRα-expressing cancerwill involve: contacting a biological sample of a subject with anFRα-specific antibody, or antigen-binding fragment thereof (such asthose derivable from the antibodies and fragments provided in Table 1),quantifying the amount of FRα present in the sample that is bound by theantibody or antigen-binding fragment thereof, comparing the amount ofFRα present in the sample to the amount of FRα determined to be in asample from the same subject at an earlier point in time; anddetermining whether the subject's FRα levels have changed over time. Atest sample with an increased amount of FRα, relative to the amountobserved for the earlier sample, may indicate progression of anFRα-expressing cancer. Conversely, a test sample with a decreased amountof FRα, relative to the amount observed for the earlier sample, mayindicate regression of an FRα-expressing cancer. Accordingly, a testsample with an insignificant difference in the amount of FRα, relativeto the amount observed for the earlier sample, may indicate a state ofstable disease for an FRα-expressing cancer. In some embodiments, theFRα levels of the sample may be compared to a known standard, alone orin addition to the FRα levels observed for a sample assessed at anearlier point in time. In some embodiments the known standard may be FRαprotein at a known concentration (e.g., a recombinant or purified FRαprotein sample). In an additional embodiment, the diagnostic method canbe followed with an additional step of administering a cancer-specifictreatment. In some embodiments the cancer-specific treatment may bedirected against FRα-expressing cancers, such as Farletuzumab.

In some embodiments the described methods involve assessing whetherFRα-expressing cancer is progressing, regressing, or remaining stable bydetermining the amount of FRα associated with a cell or tissue that ispresent in a test sample derived from the subject; and comparing theobserved amount of FRα with the amount of FRα in a sample obtained fromthe subject, in a similar manner, at an earlier point in time, wherein adifference between the amount of FRα in the test sample and the earliersample provides an indication of whether the cancer is progressing,regressing, or remaining stable. In this regard, a test sample with anincreased amount of FRα, relative to the amount observed for the earliersample, may indicate progression of an FRα-expressing cancer.Conversely, a test sample with a decreased amount of FRα, relative tothe amount observed for the earlier sample, may indicate regression ofan FRα-expressing cancer. Accordingly, a test sample with aninsignificant difference in the amount of FRα, relative to the amountobserved for the earlier sample, may indicate a state of stable diseasefor an FRα-expressing cancer. In some embodiments the amount of FRα in asample derived from the subject is assessed by contacting the samplewith an antibody that binds FRα, such as the antibodies describedherein. The sample assessed for the presence of FRα may be circulatingcells, circulating tumor cells, cells that are not tissue associated(i.e., free cells), tissues (e.g., surgically resected tumor tissue,biopsies, including fine needle aspiration), histological preparations,and the like.

In some embodiments the described methods involve assessing whetherFRα-expressing cancer is progressing, regressing, or remaining stable bydetermining the amount of FRα not associated with a cell or tissue thatis present in a test sample derived from the subject; and comparing theobserved amount of FRα with the amount of FRα in a sample obtained fromthe subject, in a similar manner, at an earlier point in time, wherein adifference between the amount of FRα in the test sample and the earliersample provides an indication of whether the cancer is progressing,regressing, or remaining stable. In this regard, a test sample with anincreased amount of FRα, relative to the amount observed for the earliersample, may indicate progression of an FRα-expressing cancer.Conversely, a test sample with a decreased amount of FRα, relative tothe amount observed for the earlier sample, may indicate regression ofan FRα-expressing cancer. Accordingly, a test sample with aninsignificant difference in the amount of FRα, relative to the amountobserved for the earlier sample, may indicate a state of stable diseasefor an FRα-expressing cancer. In some embodiments the amount of FRα in asample derived from the subject is assessed by contacting the samplewith an antibody that binds FRα, such as the antibodies describedherein. The sample assessed for the presence of FRα may be urine, blood,serum, plasma, saliva, ascites, histological preparations, and the like.

In various embodiments of the described methods, the cancer may beFRα-expressing cancer. In a particular embodiment, the FRα-expressingcancer is ovarian cancer. In some embodiments the FRα-expressing canceris endometrial cancer. In some embodiments the FRα-expressing cancer iscolorectal cancer. In some embodiments the FRα-expressing cancer isbreast cancer. In some embodiments the FRα-expressing cancer is thyroidcancer. In some embodiments the FRα-expressing cancer is fallopian tubecancer. In another embodiment, the FRα-expressing cancer is non-smallcell lung cancer, such as an adenocarcinoma.

In various aspects, the amount of FRα is determined by contacting thesample with an antibody, or antigen-binding fragment thereof, that bindsFRα. In some embodiments, the sample may be contacted by more than onetype of antibody, or antigen-binding fragment thereof, that binds FRα.In some embodiments, the sample may be contacted by a first antibody, orantigen-binding fragment thereof, that binds FRα and then contacted by asecond antibody, or antigen-binding fragment thereof, that binds FRα.Antibodies such as those described herein may be used in this capacity.For example, the antibody may be selected from among:

(a) an antibody, or antigen-binding fragment thereof, that binds thesame epitope as any one of antibody 9F3, antibody 19D4, antibody 24F12,or antibody 26B3;

(b) any one of antibody 9F3, antibody 19D4, antibody 24F12, or antibody26B3, or an antigen-binding fragment thereof;

(c) an antibody, or antigen-binding fragment thereof, that comprisesheavy chain CDR1, CDR2, and CDR3 amino acid sequences and light chainCDR1, CDR2, and CDR3 amino acid sequences of any one of antibody 9F3,antibody 19D4, antibody 24F12, or antibody 26B3, as described in Table1;

(d) an antibody, or antigen-binding fragment thereof, that comprises theheavy chain variable domain segment and light chain variable domainsegment of any one of antibody 9F3, antibody 19D4, antibody 24F12, orantibody 26B3, as described in Table 1; or

(e) an antibody having the amino acid sequence of antibody produced byany one of the cell lines deposited with the ATCC having accessionnumber PTA-11887, PTA-11884, PTA-11886, or PTA-11885, or an antigenbinding fragment thereof.

Various combinations of the antibodies and antigen-binding fragmentsdescribed in (a)-(e), as detailed above in the general sectiondescribing methods of detection, can be used to provide a “first” and“second” antibody or antigen-binding fragment to carry out the describedmonitoring methods.

In certain embodiments, the amount of FRα is determined by western blotanalysis, radioimmunoassay, immunofluorimetry, immunoprecipitation,equilibrium dialysis, immunodiffusion, electrochemiluminescence (ECL)immunoassay, immunohistochemistry, fluorescence-activated cell sorting(FACS) or ELISA assay.

Additional aspects of the summarized subject matter are provided ingreater detail in the detailed description and provided examples andassociated figures.

Kits for Detecting the FRα

Provided herein are kits for detecting FRα in a sample. These kitsinclude one of more of the FRα-specific antibodies described herein, oran antigen-binding fragment thereof, and instructions for use of thekit. In some embodiments the antibody, or antigen-binding fragment,provided in the described kits may be one or more of:

(a) an antibody, or antigen-binding fragment thereof, that binds thesame epitope as any one of antibody 9F3, antibody 19D4, antibody 24F12,or antibody 26B3;

(b) any one of antibody 9F3, antibody 19D4, antibody 24F12, or antibody26B3, or an antigen-binding fragment thereof;

(c) an antibody, or antigen-binding fragment thereof, that comprisesheavy chain CDR1, CDR2, and CDR3 amino acid sequences and light chainCDR1, CDR2, and CDR3 amino acid sequences of any one of antibody 9F3,antibody 19D4, antibody 24F12, or antibody 26B3, as described in Table1;

(d) an antibody, or antigen-binding fragment thereof, that comprises theheavy chain variable domain segment and light chain variable domainsegment of any one of antibody 9F3, antibody 19D4, antibody 24F12, orantibody 26B3, as described in Table 1; or

(e) an antibody having the amino acid sequence of antibody produced byany one of the cell lines deposited with the ATCC having accessionnumber PTA-11887, PTA-11884, PTA-11886, or PTA-11885, or an antigenbinding fragment thereof.

The provided antibody, or antigen-binding fragment, may be in solution;lyophilized; affixed to a substrate, carrier, or plate; or conjugated toa detectable label.

The described kits may also include additional components useful forperforming the methods described herein. By way of example, the kits maycomprise means for obtaining a sample from a subject, a control sample,e.g., a sample from a subject having slowly progressing cancer and/or asubject not having cancer, one or more sample compartments, and/orinstructional material which describes performance of a method of theinvention and tissue specific controls/standards.

The means for determining the level of FRα can further include, forexample, buffers or other reagents for use in an assay for determiningthe level of FRα. The instructions can be, for example, printedinstructions for performing the assay and/or instructions for evaluatingthe level of expression of FRα.

The described kits may also include means for isolating a sample from asubject. These means can comprise one or more items of equipment orreagents that can be used to obtain a fluid or tissue from a subject.The means for obtaining a sample from a subject may also comprise meansfor isolating blood components, such as serum, from a blood sample.Preferably, the kit is designed for use with a human subject.

The described kits may also include a blocking reagent that can beapplied to a sample to decrease nonspecific binding of a primary orsecondary antibody. An example of a blocking reagent is bovine serumalbumin (BSA), which may be diluted in a buffer prior to use. Othercommercial blocking reagents, such as Block Ace and ELISA Synblock (AbDserotec), Background Punisher (BIOCARE MEDICAL), and StartingBlock(Thermo Fisher Scientific) are known in the art. The described kits mayalso include a negative control primary antibody that does not bind toFRα sufficiently to yield a positive result in an antibody-baseddetection assay. In addition, the described kits may include a secondaryantibody capable of binding to a FRα primary antibody, such as antibody9F3, antibody 19D4, antibody 24F12, or antibody 26B3. In someembodiments the secondary antibody may be conjugated to a detectablelabel, such as horse radish peroxidase (HRP) or a fluorophore, to allowfor detection of the primary antibody bound to a sample. The describedkits may also include a colorimetric or chemiluminescent substrate thatallows the presence of a bound secondary antibody to be detected on asample. In some embodiments the colorimetric or chemiluminescentsubstrate may be 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)(ABTS); 3,3′,5,5′-Tetramethylbenzidine (TMB); 3,3′-Diaminobenzidine(DAB); SUPERSIGNAL™ substrate (Thermo Fisher Scientific); ECL reagent(Thermo Fisher Scientific) or other such reagents known to those ofordinary skill in the art.

The following examples are provided to supplement the prior disclosureand to provide a better understanding of the subject matter describedherein. These examples should not be considered to limit the describedsubject matter. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be apparent to personsskilled in the art and are to be included within the and can be madewithout departing from the true scope of the invention.

Example 1 Expression and Purification of Recombinant, Human FRα

To conduct the experiments associated with the studies described herein,several folate receptor alpha (FRα)-expressing cell systems or lineswere created to generate FRα-expressing cell substrates or to generatepurified recombinant human FRα protein. One expression system used wasan Sf9 insect cell line that expressed recombinant human FRα viabaculovirus. This system was prepared using a human FRα sequence,containing a leader sequence optimized for insect cell expression, aN-terminal 6× histidine (6× his) epitope tag, and the native GPIattachment site intact. The cells were then incubated in a 1 L shakeflask and log-phase cultures of Sf9 insect cells were infected with therecombinant baculovirus at a multiplicity of infection (MOI) of <1.Cells from 30 L of culture were harvested, lysed and extracted 2× with1× phosphate-buffered saline (PBS) containing 10 mM3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). TheNaCl concentration was adjusted to 300 mM and filtered through a 0.2 ummembrane. The clarified supernatant was purified by affinitychromatography, using 1×PBS with 2M NaCl, 1 mM CHAPS, pH 7.4 as washbuffer, followed by elution with 10 mM 3-(N-morpholino)propanesulfonicacid (MOPS), 3M MgCl₂, 1 mM CHAPS, pH 6.8. Peak fractions were dialyzedextensively against 1×PBS, pH 7.4, analyzed for purity by SDS-PAGE,quantitated by bicinchoninic acid assay (BCA) assay, aliquoted andstored at −80 degrees Celsius.

A Chinese hamster ovary (CHO) cell line stably expressing and secretinghuman FRα was produced using a human folate receptor alpha (FRα)sequence, containing a human immunoglobulin kappa leader sequence and aC-terminal 6× his epitope tag replacing the GPI attachment site. Onceproduced, the FRα-expressing CHO cells were grown at 25 L-scale in wavebags. To purify the secreted FRα protein, cell supernatant was clearedof cellular debris by depth filtration and then concentrated 10-fold bytangential flow filtration and diafiltered into 50 mM sodium phosphate,300 mM NaCl, 1 mM imidazole, pH 8.0. This was loaded onto a pre-packedTALON® IMAC column using an FPLC. Unbound material was washed out using50 mM sodium phosphate, 300 mM NaCl, 5 mM imidazole, pH 8.0 and boundprotein was eluted using a linear gradient of 5 mM-100 mM imidazole in50 mM sodium phosphate, 300 mM NaCl, pH 8.0. Peak fractions weredialyzed extensively against 1×PBS, pH 7.4, analyzed for purity bySDS-PAGE, quantitated by BCA assay, aliquoted and stored at −80 degreesCelsius.

A similar cell system was also produced for human folate receptor beta(FRβ), human folate receptor gamma (FRγ), and human folate receptordelta (FRδ). Briefly, constructs of either FRβ, FRγ, or FRδ containing ahuman immunoglobulin kappa leader sequence and a C-terminal 6× hisepitope tag replacing the GPI attachment site, were used to transientlytransfect 1 L cultures of 293F cells. Recombinant FR proteins werepurified as described above for human FRα.

A Chinese hamster ovary (CHO) cell line stably expressing and secretinga human mesothelin sequence, containing a human immunoglobulin kappaleader sequence and a C-terminal 6× his epitope tag replacing the GPIattachment site, was also prepared, as mesothelin served as a negativecontrol for many studies. Human mesothelin-expressing CHO cells weregrown at 25 L-scale in wave bags. To purify the secreted mesothelinprotein, cell supernatant was cleared of debris by hollow-fiberfiltration and clarified supernatant was concentrated 10-fold bytangential flow filtration. Supernatant NaCl concentration was adjustedto 300 mM NaCl and 0.5 mM imidazole. This was loaded onto a pre-packedcobalt-charged immobilized metal affinity chromatography TALON® IMACcolumn using an FPLC. Unbound material was washed out using 50 mM sodiumphosphate, 300 mM NaCl, 3 mM imidazole, pH 8.0 and bound protein waseluted using 50 mM sodium phosphate, 300 mM NaCl, 150 mM imidazole, pH8.0. Peak fractions were dialyzed extensively against 50 mM potassiumphosphate, pH 7.5. Ammonium sulfate was added to a final concentrationof 1M, and final purification was then done on a pre-packed phenylsepharose column using a step gradient of 1M-0M ammonium sulfate in 50mM potassium phosphate, pH 7.5. Peak fractions were dialyzed extensivelyagainst 1×PBS, pH 7.4, analyzed for purity by SDS-PAGE, quantitated byBCA assay, aliquoted and stored at −80 degrees Celsius.

Example 2 Production of Purified Reduced and Alkylated FRα

Efforts were undertaken to produce a reduced and alkylated antigenicform of FRα. To reduce the protein, purified FRα was concentrated to 2mg/mL in phosphate buffered saline (pH 7.4) using centrifugal filters(Amicon Ultra, 3 kD MW limit). The protein concentration was determinedusing a BCA assay (Thermo Scientific). The resultant FRα was diluted 1:1in 8M urea/PBS to generate a final concentration of 1 mg/mL FRα in PBScontaining 4M urea. Dithiothreitol solution (500 mM in PBS) was added toa final concentration of 10 mM. The solution was incubated at 65 degreesCelsius for one hour, and cooled to room temperature.

Next 1M of iodoacetamide solution in phosphate buffer saline was addedinto the reduced folate receptor solution to a final concentration of 10mM, and the reaction was kept in dark at room temperature for 30minutes. The protein remained soluble under these conditions. The finalreduced FRα to be used for immunization was stored in phosphate buffersaline containing 4M of urea, 10 mM of DTT, and 10 mM of iodoacetamide.

FIG. 1 shows the differential migration of native FRα protein and areduced and alkylated form of the protein analyzed by SDS-PAGE undernonreducing conditions.

Example 3 Production of Hybridomas Using FRα

Eight week old female Balb/c mice were immunized with hexa-histidinetagged FRα protein (n=5) or reduced and alkylated FRα protein (n=5).Initial intraperitoneal immunizations administered on day 0 comprised 50μg of the respective immunogen mixed 1:1 (v:v) with complete Freund'sadjuvant (Rockland, Cat# D614-0050). Mice were then boosted with 50 μgimmunogen mixed 1:1 (v:v) with incomplete Freund's adjuvant (Rockland,Cat# D615-0050) administered intraperitoneally 14 days later and every21 days thereafter. Blood samples were collected from immunized mice 24days after the initial immunization and every 21 days thereafter.

Collected blood samples were analyzed by direct enzyme-linkedimmunoassay (ETA) against FRα. Plates were coated with FRα protein (100ml of a 1 mg/mL solution in PBS, 0.02 M potassium phosphate, 0.15 MSodium Chloride, pH 7.2) and incubated overnight at 4° C., washed withPBS containing 0.2% polysorbate-20 (TWEEN®-20 detergent) (PBST;Rockland, Cat# MB-075-1000) and blocked with 3% fish gel (Sigma) for 1hr at room temperature. A 3-fold dilution series of individual mouseserum samples were allowed to bind for 1 hr at room temperature, plateswere then washed 3 times with PBST and subsequently probed with anHRP-conjugated rabbit-anti-mouse antibody (Rockland, Cat#610-4320) at1:2500 for 30 minutes at 37° C. TMB substrate (Rockland, Cat# TMBE-100)was added and the reaction was stopped after 30 minutes by addition of100 mL of 1M HCl prior to absorbance reading at 450 nm (MicroplateReader BENCHMARK™; Biorad). All samples were counter-screened againsthexa-histidine tagged recombinant mesothelin (mesothelin-His₆) proteinas a negative control.

Spleens from mice showing the highest antigen-specific titers wereharvested and hybridomas were prepared by electrofusion (HYBRIMUNE™Model CEEF-50B Waveform Generator; Cellectis, Romainville, France) ofsplenocytes with Sp2/0 Ag14 myeloma cells (ATTC CRL1581). Subsequently,hybridoma supernatants were screened by ELISA against FRα andrecombinant Mesothelin-His₆ as described above to select positiveparental fusion cell lines.

Selected parental cell lines determined to produce antibodies reactiveto recombinant human FRα (rhFRα) were then subcloned by limitingdilution. The antibodies produced by these cells were then retested forFRα binding and isotyped using the CLONETYPING™ System (SouthernBiotech,Birmingham, Ala.). Supernatants from these clones were further screenedby direct ELISA against three additional isoforms of the human folatereceptor (FRβ, FRγ and FRδ) to determine receptor specificity. Plateswere coated overnight with 100 μL of a 1 μg/mL solution of therespective FRα isoform at 4° C., washed with PBS containing 0.2%polysorbate-20 (TWEEN®-20 detergent) (Rockland, Cat# MB-075-1000) andblocked with 3% fish gel (Sigma). A 3-fold dilution series of culturesupernatants was allowed to bind for 1 hr at room temperature, beforeplates were washed and probed with an HRP-conjugated anti-mouse antibodyas described above. Clones producing antibodies reactive to FRβ, FRγ andFRδ were not selected for further analysis.

Four selected hybridoma clones, 19D4.B7, 26B3.F2, 24F12.B1, and9F3.H9.H3.H3.B5.G2, were deposited with the American Type CultureCollection on May 19, 2011 and were assigned ATCC accession numbersPTA-11884, PTA-11885, PTA-11886, and PTA-11887, respectively.

Example 4 Production of Purified Monoclonal Antibodies to FRα

Selected cell lines were tested for mycoplasma using a mycoplasma testkit (Rockland, Cat# MAB-012) before seeding into 1 L roller bottlescontaining serum free medium (Invitrogen, Cat#12045-076) and 5% low IgGFBS (0.1 μg/ml) (Gibco, Cat#16250-078) at 0.5×10⁵ cells/mL. Cultureswere allowed to grow at 37° C. for either 14 or 21 days, after whichsupernatant was harvested and concentrated approximately 10-fold througha 50 kDa filtration membrane (Spectrum Labs, Rancho Dominguez Calif.)and then purified using protein A chromatography (Rockland, Cat#PA50-00-0025). Bound antibody was eluted with 0.1M sodium citrate, pH3.5/4.5 depending on antibody isotype, and buffer was exchanged againstPBS by dialysis using a 12-14 kDa membranous tubing (Spectrum Labs,Rancho Dominguez Calif.). Purified antibody was sterile filtered using a0.22 nm EXPRESS™ PLUS Stericups (Millipore, Billerica Mass.) and storedat 4° C. for further testing.

Efforts were undertaken to sequence the heavy and light chains of fourselected hybridomas clones (9F3-H9, 19D4-B7, 24F12-B1, and 26B3-F2).First, total RNA was isolated from each hybridoma cell line (cellpellets of 1×10³ to 1×10⁵ cells each) using the RNAQUEOUS® kit (Ambion)according to the manufacturer's protocol. RNA was quantified using aNANODROP™ 8000 spectrophotometer (Thermo Scientific).

Isolated RNA was then amplified via multiplex RT-PCR, performed intriplicate for each hybridoma with a MASTERCYCLER® EP GradientThermocycler (Eppendorf). First, two separate gene-specific cDNAamplifications were performed for each hybridoma (≦1 ug RNA/reaction) todetermine which Ig heavy and light chain genes were used during Igrearrangement. Each cocktail consisted of unique family-specific primersdesigned to anneal to any of the potential murine Ig V gene families(ION, IgKv) and Ig constant region genes (IgHc_(Gamma), IgKc). cDNAgeneration and amplification was performed using SUPERSCRIPT® IIIOne-Step RT-PCR System with PLATINUM® Taq High Fidelity (Invitrogen)under the following conditions: 55° C. for 30 minutes and 95° C. for 2minutes, followed by 40 cycles of 95° C. for 1 minute, 55° C. for 1minute, 68° C. for 1 minute, and a final 68° C. for 10 minutescompletion step. DNA products were electrophoresed on a 2% agarose gel.Appropriate bands were excised and gel purified using the QIAQUICK® GelExtraction Kit (Qiagen) following the manufacturer's protocol. PurifiedDNA was submitted for sequencing (GENEWIZ, Inc., South Plainfield, N.J.)to determine the germline gene segments expressed by each hybridoma.

Further RT-PCR analysis suited to the particular genes identified foreach hybridoma was then performed using the same RNA source as above andgene-specific primers (in contrast to family-specific primers used inthe multiplex RT-PCR mixture). To facilitate cloning, amplified Ig cDNAswere placed into an In-Fusion (IF) expression vector, each gene-specificprimer also contained vector-compatible linker sequences which wouldenable homologous crossover. All other reagents and thermocyclerconditions are the same as those used for the multiplex RT-PCRexperiments, described above.

Example 5 Characterization of Antibody Binding to FRα

Binding characteristics of the purified monoclonal antibodies to FRαwere determined by surface plasmon resonance (SPR) experiments. All ofthe SPR experiments were performed at 25° C. using a BIACORE™ T100 withresearch grade CM5 chips (GE Healthcare), as specified by themanufacturer. Initially, anti-mouse IgG provided in the mouse antibodycapture kit (GE Healthcare) was immobilized by amide coupling to CM5sensor chips. Mouse anti-FRα monoclonal antibodies (26B3, 24F12, 19D4,or 9F3) were captured on individual flow cells per binding cycle, whilethe fourth flow cell was used as a reference. Binding experiments wereperformed with HBS-P (GE Healthcare) as running buffer and at a flowrate of 30 μL/min. Each monoclonal antibody sample (0.5 μg/mL) wasinjected for 3 minutes to capture the antibody. Various concentrationsof purified recombinant human FRα (rh-FRα) (1 nM-30 nM) were theninjected over the FRα-specific and reference surfaces for 3 minutes torecord binding sensograms using a single-cycle kinetics method. Thedissociation profile was monitored for 25 minutes. In between bindings,the surface was regenerated with a 30 μl injection of 10 mM glycine (pH1.7). The sensograms were processed and fitted to a 1:1 Langmuir bindingmodel using BIACORE™ T100 evaluation software (version 2.0.1). Some ofthe binding characteristics of antibodies 26B3, 24F12, 19D4, and 9F3 areprovided in Table 2.

TABLE 2 Binding characteristics of FRα-specific antibodies. AbbreviatedClone Name ka (1/Ms) kd (1/s) KD (M) Chi. Sq 26B3 5.24 × 10⁵ 1.43 × 10⁻⁵2.73 × 10⁻¹¹ 2.48 24F12 3.93 × 10⁵ 3.99 × 10⁻⁵ 1.02 × 10⁻¹⁰ 1.08 19D44.27 × 10⁵ 2.42 × 10⁻⁴ 5.67 × 10⁻¹⁰ 0.656 9F3 4.34 × 10⁵ 3.10 × 10⁻⁴7.15 × 10⁻¹⁰ 1.89

Example 6 Epitope Mapping of Selected FRα-Specific Antibodies

FRα-specific antibodies 26B3, 24F12, and 9F3 were further assessed inepitope binding studies using OCTET® QK system. The results showed that26B3 and 24F12, which have high affinities to purified human FRα,compete with one another for binding to FRα. Thus, these antibodies mayshare a common epitope, or have epitopes that are immediately adjacentto each other. The results also indicate that the 9F3 antibody has aunique epitope, since it did not compete with other FRα-specificantibodies for binding to FRα.

Additional epitope mapping studies were carried out by EXSAR™ usinghydrogen/deuterium exchange mass spectrometry and docking methods. Theresults of these studies for antibodies 9F3, 24F12, and 26B3 areillustrated in FIG. 2. With regard to the epitope for antibody 26B3,these data suggest that it is accessible in the native,membrane-anchored structure, given the ability of 26B3 to recognizenative FRα by flow cytometry. Furthermore, these data further suggestthat the conformational constraints of the epitope recognized by MAb26B3, as demonstrated by its inability to detect the protein on reducedwestern blots, are related to the cysteine at position 185 in the FRαprotein which forms a di-sulfide bridge with cysteine 111.

Example 7 Recognition of Denatured and Chemically-Preserved Forms of FRα

Experiments were conducted to determine whether any of the FRα-specificantibodies, described above, could recognize denatured forms of FRα. Forthese analyses CHOK1 cells stably expressing GPI-linked human FRα, β, orΔ, were lysed in 1.1% OBG buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl,1.1% OBG) supplemented with COMPLETE™ Mini Protease Inhibitor Cocktail(Roche Diagnostics, Indianapolis, Ind.) and PMSF (100 nM), and placed onice for 15 minutes. Lysates were pre-cleared by centrifugation at 13,000rpm for 15 minutes to remove debris. For reduced and denatured samples,equal amounts of protein (20 μg) were boiled for 10 minutes in NUPAGE®LDS sample buffer (Invitrogen) containing 5% β-mercaptoethanol+40 mMDTT. Proteins were separated using SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) on a 4-12% bis-tris gel (Invitrogen) andtransferred to a PVDF membrane. The membrane was blocked in PBST+5%non-fat milk for 1 h at room temperature after which time, the membranewas washed twice with PBST Immunoblotting was conducted using purifiedmouse monoclonal antibodies 9F3, 19D4, 24F12, or 26B3 (1 μg/mL) specificfor FRα, which were detected with a goat-anti-mouse HRP-conjugatedantibody and visualized using SUPERSIGNAL™ West Pico chemiluminescentsubstrate (Pierce, Rockford, Ill.). Luminescence was visualized usingthe Omega 12iC molecular imaging system (Ultra-Lum, Claremont, Calif.)with image analysis performed using ULTRAQUANT™ 6.0 software(Ultra-Lum).

Western blot analyses were also performed using purified folate receptorpreparations. For these experiments, 0.5 μg of purified human FRα, (3, For A, produced as described in Example 1, were incubated in 1×SDS-PAGEsample loading buffer (Invitrogen) with or without 20 mM DTT, boiled for10 min, and electrophoresed on 4-12% gradient SDS-PAGE gels. Protein wastransferred to PVDF membrane and blots probed as described above. Gelswere run using Benchmark™ prestained protein ladder (NOVEX®). Gels usingpurified recombinant FR proteins were also visualized via silverstainingto ensure equal amounts of protein were loaded.

Western blot analyses indicate that antibodies 19D4, 9F3, 24F12, and26B3 recognize nonreduced FRα, however, binding to reduced and denaturedsamples was not detected for any of these antibodies (FIGS. 3(A) and(B)).

Immunohistochemistry (IHC) studies were also conducted to determinewhether any of these antibodies could bind to formalin fixed paraffinembedded papillary serous ovarian cancer tissue samples. Indirect IHCtesting was performed for FRα using a MACH4™ Universal HRP-PolymerDetection Kit (Biocare Medical). Formalin-fixed paraffin-embeddedspecimens were sectioned at 5 microns on positively-charged glass slidesand heated for approximately 60 minutes at 60° C. Slides weredeparaffinized in 3 sequential baths of xylene for 3 minutes each,transferred to three sequential baths of 100% alcohol for 3 minuteseach, followed by three sequential baths of 95% alcohol for 3 minuteseach and then rinsed for 5 minutes in deionized (DI) water. Preparedsamples were then pretreated with Diva heat-induced epitope retrievalsolution (Biocare Medical) diluted to 1:10 in DI water and placed insidea pressurized decloaking chamber already filled with 500 ml of DI water.The samples were incubated for 15 minutes inside the decloaking chamber,where pressurized incubation reached a maximum of 125° C. at 16 PSI for30 seconds and then was cooled for 15 minutes down to 95° C. Slides werethen cooled at room temperature for 15 minutes. After cooling, slideswere washed in 3 sequential baths of Tris Buffered Saline/0.1%polysorbate-20 (TWEEN®-20 detergent) wash buffer (TBST) for 3 minuteseach. All subsequent buffer washes were also performed in this manner.Slides were then blocked in Peroxidase-1 (Biocare Medical) blockingsolution for 5 minutes at room temperature, washed with TBST, and thenBackground Sniper (Biocare Medical) serum-free universal blockingreagent was applied for 10 minutes at room temperature. After thesamples were blocked the slides were incubated with 2.5 μg/ml of 26B3antibody diluted in Antibody Diluent (Dako) or Universal NegativeControl—Mouse ready-to-use negative control antibody (Dako, for negativeisotype tissue) for 60 minutes at room temperature. Slides were thenwashed with TBST and incubated with MACH4™ Mouse Probe Primary AntibodyEnhancer (provided in the Biocare Medical MACH4™ kit) for 15 minutes atroom temperature. Slides were then washed again with TBST and incubatedwith a Polymer-HRP reagent (provided in the Biocare Medical MACH4™ kit)for 20 minutes at room temperature. Following incubation, slides werewashed with TBST and incubated with a 3,3′-diaminobenzidinetetrahydrochloride (DAB) solution (Dako) for 5 minutes at roomtemperature. Then slides were thoroughly rinsed with DI water 3 timesfor 30-60 seconds each and counterstained with hematoxylin (Dako) for 2minutes, washed with TBST, dehydrated in 3 sequential baths each of 95%and 100% alcohol for 30 seconds each, and cleared in 3 sequential bathsof xylene for 30 seconds each. Finally, coverslips were applied to theslides prior to analysis.

It is commonly thought that an antibody must be able to recognize alinear epitope of the antigen of interest to be effective forimmunohistochemistry of formalin fixed paraffin embedded tissue becausethe antigen is devoid of tertiary structure due to the destructivenature of fixation of tissue. Thus it was surprising that antibody 26B3was able to recognize FRα in this assay (FIG. 4), since it does notrecognize reduced and denatured FRα by western blot. Furthermore, thepattern of FRα staining with antibody 26B3 observed for normal tissueswas consistent with previously published literature using a variety ofother antibodies and techniques, with pancreas, thyroid, lung, salivarygland, kidney, hypophysis, cervix and breast showing expression tovarious degrees (Table 3). As shown in FIG. 5, the staining pattern innormal tissues, exemplified in normal lung (A) and normal kidney (B)sections, is highly restricted to epithelial cells and typically apicalin nature.

TABLE 3 FRα expression in normal human tissues Staining Tissue Type(Number/Intensity) Comments Cerebrum 0/3 Cerebellum 0/3 Adrenal 0/3Ovary 0/3 Pancreas 3/3; 2+ Limited to luminal borders of ductal andacinar cells Thyroid 2/5; 1+ (sparse) Cytoplasmic staining in follicularcells Hypophysis 3/3; 1+ Predominantly cytoplasmic Testis 0/3 Breast3/3; 1+/2+ Ductal cells with luminal and membrane staining Spleen 0/3Tonsil 0/3 Thymus 0/3 Bone Marrow 0/3 Lung 3/3; 2+ Staining in bronchialand alveolar cells Heart 0/3 Esophagus 0/3 Stomach 0/2 Small Intestine0/3 Colon 0/3 Liver 0/3 Salivary Gland 3/3; 3+ Ductal and acinar cellsKidney 3/3; 3+ Luminal staining of proximal tubular cells Prostate 0/3Endometrium 0/3 Cervix 1/3; 1+ Endocervical cells Skeletal Muscle 0/3Skin 0/3 Nerve 0/3 Mesothelium 3/3; 2+ Alveolar cells (pleura and lung)

Example 8 Recognition of Native Forms of FRα

Flow cytometry studies were conducted to assess the ability of selectedFRα-specific antibodies to bind to the native protein. For these studiesChinese hamster ovary (CHO) cells expressing FRα, were harvested,washed, and re-suspended in ice-cold growth media (RPMI supplementedwith 10% FBS). Cells were incubated for 1 hour on ice with 9F3, 19D4,24F12, or 26B3 (1 μg/mL), washed and then incubated with FITC-conjugatedsecondary antibodies [dilution 1:100] (Southern Biotech, Birmingham,Ala.). Prior to analysis, cells were labeled with 7-amino-actinomycin D(7-AAD) (BD Biosciences, Franklin Lakes, N.J.) for the exclusion ofnonviable cells. CHO cells not expressing FRα were also subjected to thesame experimental procedures, as a negative control. Cells were analyzedon an EASYCYTE™ Flow Cytometer (GUAVA® Technologies, Hayward, Calif.).The data provided in Table 4 indicate that all four antibodies arecapable of binding native FRα.

TABLE 4 FRα-specific antibodies recognize FRα expressed on the cellsurface Geometric Mean Observed for Antibody: Target 9F3 26B3 24F12 19D4Cells only 2.7 2.7 2.7 2.0 CHOK1 5.9 5.7 5.9 — FRα 759.5 853.7 777.01130.5 FRβ 6.1 5.9 6.6 — FRΔ 5.6 5.4 5.9 —

Example 9 Detection of FRα in the Serum of Subjects Known to haveOvarian Cancer

Electrochemiluminescence studies were conducted to determine whether theFRα-specific antibodies described herein could detect FRα in the serumof patients known to have ovarian cancer. For these experiments MAb 26B3was used as the capture MAb and added to ECL plates at a concentrationof 75 μg/mL. Plates were washed and 50 μL of sample serum was added toeach well and incubated for 2 hours. Serum samples were obtained fromnormal healthy females (negative control) and from ovarian cancerpatients. Samples were diluted 1:4 in PBST (phosphate buffered saline,pH7.4, containing 0.01% polysorbate-20 (TWEEN®-20 detergent)). Followingincubation, samples were washed with PBST and 25 μL/well of MAb 19D4 (1μg/mL), labeled with Ru at a ratio of approximately 13 labels/IgGmolecule, was added to each well to detect bound sample. After a 2-hourincubation period, the plates were washed with PBST and read with 2×MSDBuffer T. The results in Table 5 show that FRα in serum can be capturedand detected using monoclonal antibodies 26B3 and 19D4.

TABLE 5 Relative serum levels of FRα Category (n) Mean FRA pg/mLStandard Deviation Normal (15) 223 74 Ovarian Cancer (15) 1815 3896

Example 10 Detection of FRα in the Serum and Urine of Subjects Known tohave Ovarian Cancer

Electrochemiluminescence studies were then conducted to determinewhether the FRα-specific antibodies described herein could detect FRα inthe serum and urine of patients known to have ovarian cancer. For theseexperiments MAb 26B3 was used as the capture MAb and added to ECL platesat a concentration of 75 μg/mL. Plates were washed and 50 μL of sampleserum was added to each well and incubated for 2 hours. Matched serumand urine samples were obtained from normal healthy females (negativecontrol) and from ovarian cancer patients. Samples (serum or urine) werediluted 1:4 in PBST (phosphate buffered saline, pH7.4, containing 0.01%polysorbate-20 (TWEEN®-20 detergent)). Following incubation, sampleswere washed with PBST and 25 μL/well of MAb 19D4 (1 μg/mL), labeled withRu at a ratio of approximately 13 labels/IgG molecule, was added to eachwell to detect bound sample. After a 2-hour incubation period, theplates were washed with PBST and read with 2×MSD Buffer T. The resultsin Table 6 show that FRα in serum and urine can be captured and detectedusing monoclonal antibodies 26B3 and 19D4.

TABLE 6 Relative serum and urine levels of FRα Patient Designation SerumFRalpha pg/mL Urine FRalpha pg/mL Normal 1 398 3080 Normal 2 236 11508Normal 3 315 7704 Normal 4 320 13198 Ovarian Cancer 1 19479 368066Ovarian Cancer 2 4144 23738 Ovarian Cancer 3 986 165826 Ovarian Cancer 4719 414187

Example 11 M-Score as a Metric for Immunohistochemistry Results

A metric for staining (M-score) of each sample was developed and can bedefined as follows:

$M_{i} = {\frac{\sum\limits_{j = 1}^{3}\;{W_{j} \cdot X_{ij}}}{\sum\limits_{j = 1}^{3}\; W_{j}} = \frac{\sum\limits_{j = 1}^{3}\;{W_{j} \cdot X_{ij}}}{6}}$

In the equation, x_(ij) is the percentage of tumor stained at intensityj for patient i and w_(j) is the absolute value of the intensity(ranging from 0 to 3+). The metric has a theoretical range from zero (nopositive staining) to fifty (100% of cells staining at 3+ intensity). Assuch, the M-score is a weighted score for FRα IHC tumor cell membranestaining that captures both the proportion of FRα positive cells andstaining intensity. M-scores for each patient were averaged overmultiple tissue microarray (TMA) samples, where appropriate. If a samplewas void of results, i.e. no tumor present or necrotic tissue, theM-score was assigned to the non-void determinations.

A practical application of the above equation is presented below:

3+ 2+ 1+ 0 M Score x = 40 y = 30 z = 10 M = (3x + 2y + z)/6 3 × 40 = 1202 × 30 = 60 1 × 10 = 10 (120 + 60 + 10)/6 = 31.67Here, x=% of tumor stained with intensity 3+; y=% of tumor stained withintensity 2+; z=% of tumor stained with intensity 1+.

The positivity rate for FRα expression within a given histology wascalculated as the proportion of samples that were stained positiveaccording to the definition of a positive result (≧5% of the total tumorcells staining). Exact binomial confidence intervals were determinedusing established methods (Clopper C. J. and Pearson R. C., Biometrika.26:404-13 (1934)). Summary statistics are presented herein for alldemographic variables and for the M-score. Differences for mean valueswere determined using one-way ANOVA with post-hoc tests controlling foroverall type I error. Differences in mean values were statisticallydifferent if the p-value associated with the test was less than theBonferroni adjusted type I error for that test (maximum Type Ierror=0.05).

Example 12 Comparative Staining of Lung Carcinoma Cells with Antibody26B3 and Antibody BN3.2

There is significant variation in the literature with respect to thepercent of various carcinomas that express FRα as determined by IHC, inpart due to the use of a variety of antibodies, most of which are notcommercially available. One FRα specific MAb that is commerciallyavailable and has been demonstrated to detect FRα on FFPE sections byIHC is antibody BN3.2 (Leica Microsystems, Buffalo Grove, Ill.).Therefore studies were conducted to compare antibody BN3.2 to antibody26B3 for both specificity and sensitivity for the detection of FRα usinga commercial TMA containing various histological types of lung cancer.Both antibodies were highly specific for adenocarcinoma as compared withother histologic subtypes, particularly squamous cell carcinoma.However, antibody 26B3 was significantly more sensitive than BN3.2,identifying 26/36 (72%; M-score mean±SD=19.84±18.64) and 22/36 (61%;M-score mean±SD=11.38±14.25) adenocarcinoma samples, respectively(p<0.0001). These data demonstrate that antibody BN3.2 is significantlyless sensitive than antibody 26B3 for detecting FRα expression on FFPEtissue samples and, as shown in FIG. 6, the relationship in observedM-scores on lung adenocarcinoma samples for these two antibodies isnon-linear.

Example 13 Detection of FRα in Subjects Known to have Adenocarcinoma ofthe Lung

Experiments were conducted to determine whether the presence of FRαpositive histology, as detected by antibody 26B3, was associated withparticular forms of lung cancer. A tissue microarray having duplicatesamples of normal and cancerous, stage I, stage II, stage III, and stageIV, lung tissue specimens was assessed for FRα expression via IHCstaining using antibody 26B3, as described in Example 7. As can be seenfrom the data in Table 6, FRα is associated with adenocarcinomasrelative to squamous cell carcinomas, which exhibited limited positivestaining.

TABLE 7 Histological evaluation of cancerous tissue samples MembranePositive Membrane Staining Negative Positive Total Histology GroupsAdenocarcinoma Count 11 27 38 % within Histology Groups 28.9% 71.1%100.0% Squamous Count 28 3 31 % within Histology Groups 90.3%  9.7%100.0% Other Carcinomas Count 17 4 21 % within Histology Groups 81.0%19.0% 100.0% Normal Count 2 8 10 % within Histology Groups 20.0% 80.0%100.0% Total Count 58 42 100 % within Histology Groups 58.0% 42.0%100.0%

Further analyses were performed on 89 of the histological samples in thetissue microarray where 36 (40%) were adenocarcinoma, 32 (36%) weresquamous cell carcinoma, 2 (2%) were adenosquamous carcinomas, and theremaining 19 (21%) represented a variety of histologies (Table 8). Theoverall rates of FRα positivity varied substantially for each of thehistologic subtypes. A significantly higher proportion of adenocarcinomatumors were positive for FRα when compared to squamous cell carcinomas(72% versus 13%, p<0.0001). Of the 4 positive squamous cell carcinomasamples, only 1 showed 3+ staining on both samples; 1 had intermediate(2+) staining on both samples and the other 2 were very weakly positivein a single sample (5-10% of tumor cells at 1+). Furthermore, the twoadenosquamous carcinoma samples were also shown to be positive for FRα,with staining restricted to the adenocarcinoma portion of these samples(FIG. 7).

TABLE 8 Distribution of FRα Expression Across NSCLC Type# FRα FRαnegative positive Variable N (%) N (%) Total P value* Tumor HistologyNormal 1 (10%)  9 (90%) 10 Squamous cell carcinoma 28 (87%)   4 (14%) 32<0.0001 Large cell carcinoma 3 (60%)  2 (40%) 5 Small cell carcinoma 7(87%)  1 (13%) 8 Neuroendocrine carcinoma 4 (67%)  2 (33%) 6Adenocarcinoma** 10 (16%)  28 (74%) 38 Tumor Grade Grade 1 1 (20%)  4(80%) 5 Grade 2 5 (22%) 18 (78%) 23 Grade 3 4 (40%)  6 (60%) 10 0.517Tumor Stage Stage I 4 (29%) 11 (71%) 15 Stage II 2 (17%) 10 (83%) 12Stage III + IV*** 4 (36)    7 (64)  11 0.563 Gender Female 3 (18%) 14(82%) 17 Male 7 (33%) 14 (67%) 21 0.46 #US Biomax Lung Cancer TMA(catalog # BC041114; 90 cases, duplicate cores) *P values determinedusing Fisher's exact test or chi-square test: squamous cell carcinomaversus adenocarcinoma p < 0.0001; males versus female, p = 0.46; stage,p = 0.563; grade, p = 0.517 **Includes 2 adenosquamous cases, bothpositive for FRα in the adenocarcinoma portion only ***Only 1 stage IVcase

M-score analyses of duplicate adenocarcinoma histology samples showedlittle variation in staining by antibody 26B3 (FIG. 8), a reflection ofthe robustness of antibody 26B3 staining. Also, an examination ofM-scores by stage and grade within the adenocarcinoma histologic subtypeindicated that neither stage nor grade of disease was associated withthe degree of staining as defined by the M-scores (data not shown).

The M-score distribution for FRα staining of lung adenocarcinoma andsquamous cell carcinoma samples is shown in FIG. 9. The mean (±SD)M-scores for adenocarcinoma and squamous cell carcinoma samples stainedwith antibody 26B3 were 19.84 (±18.64) and 1.39 (±5.54), respectively(p<0.0001). The M-score for adenocarcinoma was also significantly higherwhen compared against all other lung cancer histologic types. Inaddition, a Tree Analysis was performed to determine the odds for thehistology of the cancer being adenocarcinoma. An M-score>21.7 resultedin an odds ratio (OR) of 16, further demonstrating that FRα ispredominately expressed in the adenocarcinoma histology (analysis notshown).

Formalin-fixed, paraffin-embedded (FFPE) tissue blocks are rarelyavailable from patients diagnosed with late stage lung cancer, assurgical resections are not typically performed. Therefore, studies wereperformed to determine the suitability of fine needle aspirate (FNA)specimens for FRα IHC using MAb 26B3, as late stage lung cancer is mostfrequently diagnosed via small biopsy or cytology material. For thesestudies, samples were obtained from nine late-stage adenocarcinomapatients diagnosed by cytological evaluation of a thoracic lymph nodeaspirate (FIG. 10) and demonstrated that the rate of FRα positivity(63%) was comparable to that seen for the histological specimensassessed on the lung cancer TMA. Although only a small sample size,these data suggest that cytologic specimens may be a suitable tissuesource for determining FRα expression in late stage adenocarcinomapatients.

Example 14 FRα is Expressed by CK+/CD45− Cells, but not CK−/CD45+Cells,Isolated from the Blood of Patients Known to have Non-Small Cell LungCarcinoma

Studies were conducted to determine the expression profile of FRα oncirculating tumor cells (CTCs) of Patients known to have Non-Small CellLung Carcinoma. For these studies, blood samples were obtained from 15healthy donors and 5 stage IV lung cancer patients and then enriched forCTCs via dielectrophoresis field flow fractionation using ApoCell'sAPOSTREAM® system. After enrichment, each sample was stained forcytokeratin (CK), CD45 (protein tyrosine phosphatase receptor type C),nuclei, and FRα. FRα staining was performed using antibody 26B3 as theprimary antibody, which was then detected using a mouse-specific,secondary antibody conjugated to DYLIGHT® 649 fluorescent dye. As shownin Table 9, FRα expression was observed by CK+/CD45− CTCs, but notCK−/CD45+CTCs.

TABLE 9 Expression of FRα by circulating tumor cells of patients knownto have non-small cell lung carcinoma CK−/CD45+/ FRα MFI Patient IDCK−/CD45+ count FRα+ % (CK−/CD45+) Patient 1 2,270 0.0 NA Patient 224,462 0.0 NA Patient 3 26,503 0.0 NA Patient 4 16,540 0.0 NA Patient 52,652 0.0 NA CK+/CD45− cell count CK+/CD45−/ FRα MFI Patient ID in 7.5mL of blood FRα+ % (CK+/CD45−) Patient 1 55 15.6 82,195 Patient 2 10532.8 172,669 Patient 3 216 9.3 146,521 Patient 4 57 16.7 179,027 Patient5 47 8.1 277,335

Example 15 5-Year Survivorship of Subjects with and withoutFRα-Expressing Adenocarcinoma of the Lung

Experiments were conducted to determine whether the presence of FRαpositive histology, as detected by antibody 26B3, could be associatedwith either improved or diminished 5-year survivorship. Normal andcancerous, stage I or stage II adenocarcinoma, lung tissue specimenswere subjected to IHC staining, as described in Example 7, and thenread. The percentage of 3+, 2+, 1+ and 0 intensity of the stain on thetumor was recorded. There were 177 slides that were interpretable asduplicate or triplicate of a patient. When combined with the evaluableclinical and histological data 53 evaluable cases were identified. Theanalyses were performed in view of data relating to demographic,clinical and survival status at 5 years past diagnosis of non-small celladenocarcinoma of the lung.

To determine an optimal cut-point for the M-score a receiver operatingcharacteristic (ROC) analysis was performed. Diagnostic accuracy was ofno importance in this analysis; however, the ratio of the diagnosticlikelihood ratio of the positive test to the diagnostic likelihood ratioof the negative test was important. These ratios are defined asdescribed in Pepe M S, The statistical evaluation of medical tests forclassification and prediction, New York: Oxford University Press (2003).At a cut-point of 10 the odds ratio achieved a maximum of 6.62. Thisvalue of M was chosen to determine the positivity of a stained slide.

Kaplan-Maier survival functions were produced with FRα association asthe prognostic factor. A log rank test indicated that being positive forFRα was beneficial for non-fatal events (Chi-sq=7.34, df=1, p=0.007).FIG. 11 illustrates the survival functions for stage I and stage IIadenocarcinoma groups deemed to be FRα positive and FRα negative by 26B3detection. At 5 years the hazard ratio is 2.42. This indicates thatsubjects having tumors that are negative (M<10) for FRα are 2.5 timesmore likely to die within five years of diagnosis than subjects withFRα-positive tumors (M≧10).

Example 16 Folate Receptor Alpha Expression is Associated withTriple-Negative Forms of Breast Cancer

Studies were conducted to assess the expression of FRα by breast cancertissue samples. Analyses were conducted using tissue microarray (TMA)samples stained with antibody 26B3 as described in Example 7 and FFPEhistology samples prepared and stained with antibody 26B3 as describedin Example 7.

The distribution of histologies present on the breast cancer TMA (U.S.BioMAX catalog # BR1503a; 72 cases, duplicate cores) are shown in Table10, the majority of the cases represented being identified as invasiveductal carcinoma (IDC). The TMA included 2 normal breast samples, whichwere positive for FRα expression as determined by MAb 26B3. Staining innormal breast was restricted to ductal cells with luminal and membranestaining Two of three fibroadenoma cases (67%), 0/2 cystosarcoma cases(0%) and 1/6 ductal carcinoma in situ cases (17%) were positive for FRα.The single invasive lobular carcinoma (ILC) was negative for FRαstaining. Of the 59 IDC samples 18 (31%) were positive for FRα (FIG.12). Given the small number of positive cases on this TMA a validanalysis of FRα expression relative to stage or grade was not possible;however, it should be noted that the majority of samples were either T1or T2. FRα expression was shown to associate with ER/PR negative tumorsrelative to ER/PR positive tumors (p=0.012) and with triple negativebreast cancers (TNBC) (ER/PR+ or Her2+ versus ER/PR/Her2−, p<0.0001).

Of the 18 FRα positive IDC cases, only 2 (11%) were Her2 positivemeaning that the vast majority (89%) were Her2 negative. These datasuggest that FRα positivity tracks more closely with Her2 negativity.Further, of the 18 FRα positive IDC cases, 3 were estrogen receptor (ER)positive and 4 were progesterone receptor (PR) positive, but all ER/PRpositive/FRα positive cases were Her2 negative. Of the FRα positive IDCcases 12/18 (67%) were triple negative breast cancers (TNBC), suggestingthat FRα may be a marker and target for very poor prognosis TNBCmolecular subtype. Looking at the TMA as a whole, only 2/13 (15%) of allHer2 positive cases were also positive for FRα while 16/46 (35%) of theHer2 negative cases were also FRα positive, supporting the suggestionthat FRα expression correlates negatively with Her2 expression. Arepresentation of the distribution of M-scores for this TMA relative tomolecular subtype (her-2 (+) and her-2 (−)) is shown in FIG. 13.

The TMA described above was composed primarily of early stage breastcancers: stage I, 6/60 (10%); stage II, 44/60 (73%); stage III, 10/60(17%). Therefore, to confirm and extend the results obtained on the TMA,61 FFPE tissue blocks from stage IV(T4) Her2 negative breast cancerswith known ER/PR expression ranging from 0-100% positive were assessed(FFPE tissue blocks were obtained from the archives of GenzymeGenetics). All 61 of these samples were from metastases, not the primarytumor. The results of this study are summarized in Table 11.

TABLE 10 Distribution of FRα positivity across histology types-TMA dataFRα positive FRα negative Tumor Histology N (%) N (%) Total P value*Normal  2 (100%) 0 (0%) 2 Fibroadenoma  2 (67%)  1 (33%) 3 Cystosarcoma0 (0%)  2 (100%) 2 DCIS—Ductal carcinoma in situ  1 (17%)  5 (83%) 6ILC—Invasive lobular 0 (0%)  1 (100%) 1 carcinoma IDC—Invasive ductalcarcinoma 18 (31%) 41 (69%) 59 Total carcinomas: 21 (30%) 50 (70%) 71IDC Molecular subtype analysis: ER/PR+  4 (14%) 24 (86%) 28 {closeoversize bracket} 0.012 ER/PR− 14 (45%) 17 (55%) 31 Her2+  2 (15%) 11(85%) 13 {close oversize bracket} 0.307 Her2− 16 (35%) 30 (65%) 46ER/PR/Her2− 12 (67%)  6 (33%) 18 <0.0001 (ER/PR+ or Her2+ versusER/PR/Her2−) T1  3 (43%)  4 (57%) 7 T2 10 (26%) 29 (74%) 39 T3  5 (63%) 3 (37%) 8 T4 0 (0%)  5 (100%) 5 N0 18 (35%) 33 (65%) 51 {close oversizebracket} 0.092 N1/N2** 0 (0%)  8 (100%) 8 Grade 1  1 (14%)  6 (86%) 7{close oversize bracket} 0.393 Grade 2 12 (36%) 21 (64%) 33 {closeoversize bracket} 0.6465 Grade 3  5 (26%) 14 (74%) 19 *P valuescalculated via 2X2 contingency table analysis using Fisher's exact test.**4/8 (50%) of N1/N2 samples were Her2+

FRα expression (FIG. 14) was found in 22/61 (36%) of these patients,demonstrating that the percent of FRα positive specimens/tumorsdetermined in early stage disease is retained in late stage metastaticdisease in a Her2 negative population (TMA positivity=35%; stage IVmetastatic disease=36%). Of the 22 FRα positive stage IV metastaticpatients, only 3 (14%) showed any positivity for ER/PR with suchpositivity trending in the low range (up to 30%). As such, 19/22 (86%)FRα positive patients were of the triple negative molecular subtype.Again, these data compare favorably with the data obtained in earlystage disease on the TMA where 67% of all FRα positive patients were ofthe triple negative subtype.

TABLE 11 Distribution of FRα positivity in molecular subtypes ofmetastatic breast cancer samples Tumor FRα FRα Molecular positivenegative subtype N (%) N (%) Total P value* Total Samples: 22 (36%) 39(64%) 61 ER/PR+  3 (14%) 20 (86%) 23 ER/PR/Her2− 19 (50%) 19 (50%) 380.0054 (ER/PR+ versus ER/PR/Her2−) Grade 1  3 (30%)  7 (70%) 10 Grade 211 (28%) 28 (72%) 39 1.0 (Grade 1 versus Grade 2) Grade 3  8 (67%)  4(33%) 12 0.037 (Grade 1 or 2 versus Grade 3) *P values calculated via2X2 contingency table analysis using Fisher's exact test.

Additionally, samples from stage IV metastatic disease were obtainedfrom a number of metastatic sites including lymph node, bone, skin andliver as well as fluid and fine needle aspirate (FNA) samples obtainedprimarily from pleura and paracentesis. Several of these ‘fluidbiopsies’ stained positive for FRα (FIG. 15) suggesting the generalapplicability of the described IHC methodology to multiple samplestypes.

Example 17 Evaluation of Histological Gynecologic Cancer Samples forExpression of Folate Receptor Alpha

Immunohistochemical studies were conducted to evaluate FRα expression ingynecologic malignancies involving ovary, endometrium and fallopiantube. Analyses were conducted using tissue microarray (TMA) samplesstained with antibody 26B3 as described in Example 7 and FFPE histologysamples prepared and stained with antibody 26B3 as described in Example7. Commercial tissue microarrays were obtained from US Biomax, Inc.(Rockville, Md.) for ovarian carcinomas (catalog # OV1921; 96 cases,duplicate cores); endometrial carcinomas (catalog # EMC1021; 102 cases,single cores); and fallopian tube carcinomas (catalog # UTE601; 30cases, duplicate cores).

A sample was considered positive for FRα expression if the percentage ofthe tumor cells positive for membranous staining was greater than orequal to 5% at any intensity. A sample was rejected and therefore notincluded in the analyses if the gynecologic pathologist determined itwas either missing entirely or was composed of necrotic tissue with aninsufficient number of viable cells for evaluation. Of the endometrialsamples, six contained only atypical complex hyperplasia withoutadenocarcinoma. Histologic classification of cell type and grade werebased the WHO Classification of Breast and Female Genital Organs(Tavassoli and Devilee). Clinical stage based on FIGO and TNM system wasprovided by the manufacturer of the TMA (US Biomax).

The positivity rate for FRα expression within a given tumor type wascalculated as the proportion of tumors that were stained positiveaccording to the definition of a positive result (±5% tumor cellmembrane staining). Differences in FRα positivity between groups, e.g.histologies, stage or grade, were assessed using 2×2 contingency tablesand Fisher's exact test. Differences in mean values were statisticallydifferent if the p-value associated with the test was less than theBonferroni adjusted type I error for that test (maximum Type Ierror=0.05).

Membrane and cytoplasmic staining intensity was scored as 0, nostaining; 1+, weak; 2+, moderate and 3+, strong. The percent of cellsfor each intensity in the sample was also determined Tissue was analyzedunder 4×, 10×, 20× and 40× objectives. Strong membrane staining (3+) wasreadily visualized under 4× and confirmed at 10×. Moderate membranestaining (2+) was visible at 10× and confirmed at 20×. Weak staining(1+) required 20× or 40× (FIG. 16). In the presence of 3+ staining, themembrane was thick occurring at apical and lateral cell borders. Intangential sections, a complete circumferential pattern was evident(FIGS. 16(A) and (B)). 2+ membrane staining was weaker in intensity andthinner than 3+, usually localized on the apical luminal borders andoccasionally on lateral cell borders. 1+ weak membrane was generallylimited to the luminal borders. The accompanying cytoplasmic stainingwas variable, depending on the type of tumors.

Of the 94 evaluable samples on the ovarian tumor TMA, 70 (74%) were ofthe serous type, 10 (11%) were mucinous, 4 (4%) endometrioid, 3 (3%)clear cell type and the remaining 7 (8%) were miscellaneous rare tumors.Of the 87 samples of ovarian carcinomas, the FRα positive rate for eachcell type was as follows: 100% (70/70) in serous type, 80% (8/10)mucinous type, 75% (3/4) endometrioid type, and 67% (2/3) clear celltype. The difference between serous and mucinous type is significantwith p value at 0.014 by Fisher's exact test (Table 12). FRα status wasnot significant for histologic grade or clinical stage. Co-existingcytoplasmic staining was usually 2+ or 3+ in serous type and weaker andless frequent in other tumor types.

TABLE 12 Distribution of FRα positivity in relation to histology type,clinical stage and histologic grade in ovarian carcinomas FRα negativeFRα positive Tumor Histology* N (%) N (%) Total P value** Serouscarcinoma 0 (0%)  70 (100%) 70 Mucinous carcinoma  2 (20%)  8 (80%) 100.014 Endometrioid carcinoma  1 (25%)  3 (75%) 4 Clear cell carcinoma  1(33%)  2 (67%) 3 Total 4 (5%) 83 (95%) 87 Stage II 4 (9%) 41 (91%) 45Stage III 0 (0%)  29 (100%) 29 0.15 Stage IV 0 (0%)  13 (100%) 13 Grade1  2 (15%) 11 (85%) 13 NS*** Grade 2 1 (3%) 31 (97%) 32 NS    Grade 3 1(3%) 39 (97%) 40 NS    *1 transitional cell carcinoma, 1 squamouscarcinoma, 1 Embryonal carcinoma, 2 yolk sac tumors and 2 granulosa celltumors not included in analysis **P values determined using Fisher'sexact test or chi-square test: serous carcinoma versus mucinouscarcinoma p = 0.014 ***NS = Not Significant

In the endometrial samples, FRα was expressed in 80% (4/5) of normal(FIG. 17(A)), 100% (6/6) of atypical complex hyperplasia (FIG. 17(B)),and 89% (80/90) of adenocarcinomas, including 88 endometrioid type and 1clear cell type (FIGS. 18 and 19). Eight endometrioid adenocarcinomascontained areas of squamous metaplasia. In the normal endometrium,membrane staining was weak and limited to the apical luminal borders(FIG. 17(A)). In atypical complex hyperplasia and carcinomas, stainingwas predominantly luminal with additional staining on lateral cellborders in some cases (FIG. 17(B)). In the presence of 3+ membranestaining, cytoplasmic staining varied in intensity from strong (FIG.18(A)) to weak (FIGS. 18(B) and (C)). Tumor cells with 1+ or 2+ membranestaining rarely expressed cytoplasmic staining. The majority ofmetaplastic squamous cells and clear cells exhibited moderate to strongmembrane staining (FIGS. 19(A) and (B)).

FRα expression was positive in 100% of grade 1, 96% of grade 2 and 74%of grade 3 tumors (grade 1 vs. grade 3, p value=0.0029; grade 2 vs.grade 3, p=0.034). FRα status was not significant in relation to T1 vs.T2/3; NO vs. N1, and stage I vs. stage II/III.

Seventeen cores of normal fallopian tubes, 16 samples of chronicsalpingitis and 20 tubal serous carcinomas were all strongly positivefor membrane and cytoplasmic staining (FIGS. 20(A) to (C)).

Example 18 Evaluation of Histological Colorectal Samples for Expressionof Folate Receptor Alpha

Immunohistochemical studies were conducted to evaluate FRα expression incolorectal tissue samples. Analyses were conducted using tissuemicroarray (TMA) samples obtained from US Biomax (catalog # BC051111).The TMA contained 90 duplicate samples of tissues obtained from subjectsknown to have colorectal cancer and 10 normal colorectal samples. Thesamples were stained with antibody 26B3 as described in Example 7. Ofthe 90 samples obtained from subjects known to have colorectal cancer,18 (20%) were positive for FRα expression, while none of the normalsamples were positive. In addition, positive stating was generallymedium to weak and no apparent relationship to stage of disease wasdiscernible.

Example 19 Evaluation of Histological Thyroid Samples for Expression ofFolate Receptor Alpha

Immunohistochemical studies were conducted to evaluate FRα expression inthyroid tissue samples. Analyses were conducted using tissue microarray(TMA) samples obtained from US Biomax (catalog # TH802a). The sampleswere stained with antibody 26B3 as described in Example 7. Thyroidpapillary carcinoma was strongly positive for FRα membrane expression(26/28, 93%) and was distinguishable from medullary carcinoma, where all5 samples were negative for FRα staining, in agreement with previousreports. Interestingly, follicular adenomas were separable intomacrofollicular type and microfollicular type with 3/13 (23%) and 18/22(82%) showing positivity for FRα expression, respectively. Somepositivity was also seen in the small number of Hurthle cell tumors(2/3, 67%) and follicular carcinoma (3/7, 43%) samples on this TMA.These results are summarized in Table 13.

TABLE 13 Expression of FRα in thyroid tissue samples Thyroid CancerHisotologic FRα positive FRα negative Subtype (N = 78) N (%) N (%)Papillary carcinoma, 28 (36%) 26 (93%) 2 (7%) Medullary carcinoma, 5(6%) 0 (0%)  5 (100%) Follicular Adenoma, macrofollicular  3 (23%) 10(77%) type, 13 (17%) Follicular Adenoma, microfollicular 18 (82%)  4(18%) type, 22 (28%) Hurthle cell tumor, 3 (4%)  2 (67%)  1 (33%)Follicular carcinoma, 7 (9%)  3 (43%)  4 (57%)

What is claimed is:
 1. A method of detecting folate receptor alpha (FRα)in a biological sample, comprising exposing the sample to an antibody orantigen-binding fragment comprising: a) a light chain CDR1 having theamino acid sequence of SEQ ID NO: 2, a light chain CDR2 having the aminoacid sequence of SEQ ID NO: 3, a light chain CDR3 having the amino acidsequence of SEQ ID NO: 4, a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 6, a heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 7, and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 8; b) a light chain CDR1 having the amino acidsequence of SEQ ID NO: 10, a light chain CDR2 having the amino acidsequence of SEQ ID NO: 11, a light chain CDR3 having the amino acidsequence of SEQ ID NO: 12, a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 14, a heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 15, and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 16; c) a light chain CDR1 having the amino acidsequence of SEQ ID NO: 18, a light chain CDR2 having the amino acidsequence of SEQ ID NO: 19, a light chain CDR3 having the amino acidsequence of SEQ ID NO: 20, a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 22, a heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 23, and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 24; or d) a light chain CDR1 having the aminoacid sequence of SEQ ID NO: 26, a light chain CDR2 having the amino acidsequence of SEQ ID NO: 27, a light chain CDR3 having the amino acidsequence of SEQ ID NO: 28, a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 30, a heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 31, and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 32; and detecting folate receptor alpha (FRα). 2.The method of claim 1, wherein the biological sample is derived fromurine, blood, serum, plasma, saliva, ascites, circulating cells,circulating tumor cells, cells that are not tissue associated, tissues,surgically resected tumor tissue, biopsies, fine needle aspirationsamples, or histological preparations.
 3. A method of diagnosing afolate receptor alpha-expressing cancer in a subject, wherein the folatereceptor alpha-expressing cancer is folate receptor alpha-expressinglung cancer, folate receptor alpha-expressing ovarian cancer, folatereceptor alpha-expressing breast cancer, folate receptoralpha-expressing thyroid cancer, or folate receptor alpha-expressingcolorectal cancer, comprising: a) exposing a biological sample of thesubject to an antibody, or antigen-binding fragment thereof, comprising:a light chain CDR1 having the amino acid sequence of SEQ ID NO: 2, alight chain CDR2 having the amino acid sequence of SEQ ID NO: 3, a lightchain CDR3 having the amino acid sequence of SEQ ID NO: 4, a heavy chainCDR1 having the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 7, and a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 8; a light chain CDR1having the amino acid sequence of SEQ ID NO: 10, a light chain CDR2having the amino acid sequence of SEQ ID NO: 11, a light chain CDR3having the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 14, a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 15, and a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 16; a light chain CDR1having the amino acid sequence of SEQ ID NO: 18, a light chain CDR2having the amino acid sequence of SEQ ID NO: 19, a light chain CDR3having the amino acid sequence of SEQ ID NO: 20, a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 22, a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 23, and a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 24; or a light chain CDR1having the amino acid sequence of SEQ ID NO: 26, a light chain CDR2having the amino acid sequence of SEQ ID NO: 27, a light chain CDR3having the amino acid sequence of SEQ ID NO: 28, a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 30, a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 31, and a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 32; b) quantifying theamount of folate receptor alpha (FRα) present in the sample; and c)comparing the amount of folate receptor alpha (FRα) present in thesample to a known standard, wherein the known standard comprises a FRαlevel observed in subjects known not to have the cancer or a FRα levelobserved in subjects known to have the FRα-expressing cancer, wherein anincrease in the amount of FRα in the biological sample of the subjectrelative to the FRα level observed in subjects known not to have thecancer or a similar amount of FRα in the biological sample of thesubject relative to the FRα level observed in subjects known to have thecancer is indicative of the presence of the cancer.
 4. The method ofclaim 3, wherein the biological sample is derived from urine, blood,serum, plasma, saliva, ascites, circulating cells, circulating tumorcells, cells that are not tissue associated, tissues, surgicallyresected tumor tissue, biopsies, fine needle aspiration samples, orhistological preparations.
 5. The method of claim 3 wherein the step ofquantifying the amount of folate receptor alpha (FRα) present in thesample comprises western blot analysis, radioimmunoassay,immunofluorimetry, immunoprecipitation, equilibrium dialysis,immunodiffusion, electrochemiluminescence (ECL) immunoassay,immunohistochemistry, fluorescence-activated cell sorting (FACS) orELISA assay.
 6. A method of monitoring a folate receptoralpha-expressing cancer in a subject, wherein the folate receptoralpha-expressing cancer is folate receptor alpha-expressing lung cancer,folate receptor alpha-expressing ovarian cancer, folate receptoralpha-expressing breast cancer, folate receptor alpha-expressing thyroidcancer, or folate receptor alpha-expressing colorectal cancer,comprising: a) exposing a test biological sample of the subject to anantibody, or antigen-binding fragment thereof, comprising: a light chainCDR1 having the amino acid sequence of SEQ ID NO: 2, a light chain CDR2having the amino acid sequence of SEQ ID NO: 3, a light chain CDR3having the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 7, and a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 8; a light chain CDR1having the amino acid sequence of SEQ ID NO: 10, a light chain CDR2having the amino acid sequence of SEQ ID NO: 11, a light chain CDR3having the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 14, a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 15, and a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 16; a light chain CDR1having the amino acid sequence of SEQ ID NO: 18, a light chain CDR2having the amino acid sequence of SEQ ID NO: 19, a light chain CDR3having the amino acid sequence of SEQ ID NO: 20, a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 22, a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 23, and a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 24; or a light chain CDR1having the amino acid sequence of SEQ ID NO: 26, a light chain CDR2having the amino acid sequence of SEQ ID NO: 27, a light chain CDR3having the amino acid sequence of SEQ ID NO: 28, a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 30, a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 31, and a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 32; b) quantifying theamount of folate receptor alpha (FRα) present in the sample that isbound by the antibody or antigen-binding fragment thereof; and c)comparing the amount of folate receptor alpha (FRα) present in thesample to a biological sample obtained from the subject at an earlierpoint in time; wherein an increase in the amount of FRα in the testbiological sample relative to the amount of FRα observed in thebiological sample obtained from the subject at the earlier point in timeis indicative of progression of the FRα-expressing cancer, a decrease inthe amount of FRα in the test biological sample relative to the amountof FRα observed in the biological sample obtained from the subject atthe earlier point in time is indicative of regression of theFRα-expressing cancer, and an insignificant difference in the amount ofFRα in the test biological sample relative to the amount of FRα observedin the biological sample obtained from the subject at the earlier pointin time is indicative of stable FRα-expressing cancer.
 7. The method ofclaim 6, wherein the biological sample is derived from urine, blood,serum, plasma, saliva, ascites, circulating cells, circulating tumorcells, cells that are not tissue associated, tissues, surgicallyresected tumor tissue, biopsies, fine needle aspiration samples, orhistological preparations.
 8. The method of claim 6 wherein the step ofquantifying the amount of folate receptor alpha (FRα) present in thesample comprises western blot analysis, radioimmunoassay,immunofluorimetry, immunoprecipitation, equilibrium dialysis,immunodiffusion, electrochemiluminescence (ECL) immunoassay,immunohistochemistry, fluorescence-activated cell sorting (FACS) orELISA assay.
 9. A method of treating folate receptor alpha-expressingcancer in a subject, wherein the folate receptor alpha-expressing canceris folate receptor alpha-expressing lung cancer, folate receptoralpha-expressing ovarian cancer, folate receptor alpha-expressing breastcancer, folate receptor alpha-expressing thyroid cancer, or folatereceptor alpha-expressing colorectal cancer, comprising: a) exposing abiological sample of the subject to an antibody, or antigen-bindingfragment thereof, comprising: a light chain CDR1 having the amino acidsequence of SEQ ID NO: 2, a light chain CDR2 having the amino acidsequence of SEQ ID NO: 3, a light chain CDR3 having the amino acidsequence of SEQ ID NO: 4, a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 6, a heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 7, and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 8; a light chain CDR1 having the amino acidsequence of SEQ ID NO: 10, a light chain CDR2 having the amino acidsequence of SEQ ID NO: 11, a light chain CDR3 having the amino acidsequence of SEQ ID NO: 12, a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 14, a heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 15, and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 16; a light chain CDR1 having the amino acidsequence of SEQ ID NO: 18, a light chain CDR2 having the amino acidsequence of SEQ ID NO: 19, a light chain CDR3 having the amino acidsequence of SEQ ID NO: 20, a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 22, a heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 23, and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 24; or a light chain CDR1 having the amino acidsequence of SEQ ID NO: 26, a light chain CDR2 having the amino acidsequence of SEQ ID NO: 27, a light chain CDR3 having the amino acidsequence of SEQ ID NO: 28, a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 30, a heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 31, and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 32; b) quantifying the amount of folate receptoralpha (FRα) present in the sample; c) comparing the amount of folatereceptor alpha (FRα) present in the sample to a known standard, whereinthe known standard comprises a FRα level observed in subjects known notto have the cancer or a FRα level observed in subjects known to have theFRα-expressing cancer, wherein an increase in the amount of FRα in thebiological sample of the subject relative to the FRα level observed insubjects known not to have the cancer or a similar amount of FRα in thebiological sample of the subject relative to the FRα level observed insubjects known to have the cancer is indicative of the presence of thecancer; and d) administering to the subject, or prescribing, a treatmentfor the cancer if the FRα level in the biological sample of the subjectis indicative of the presence of the cancer.
 10. The method of claim 9,wherein the biological sample is derived from urine, blood, serum,plasma, saliva, ascites, circulating cells, circulating tumor cells,cells that are not tissue associated, tissues, surgically resected tumortissue, biopsies, fine needle aspiration samples, or histologicalpreparations.
 11. The method of claim 9 wherein the step of quantifyingthe amount of folate receptor alpha (FRα) present in the samplecomprises western blot analysis, radioimmunoassay, immunofluorimetry,immunoprecipitation, equilibrium dialysis, immunodiffusion,electrochemiluminescence (ECL) immunoassay, immunohistochemistry,fluorescence-activated cell sorting (FACS) or ELISA assay.
 12. Themethod of claim 9 wherein said treatment comprises farletuzumab.
 13. Amethod of detecting folate receptor alpha (FRα) in a biological sample,comprising exposing a biological sample of a subject to a firstantibody, or antigen-binding fragment thereof, comprising: a light chainCDR1 having the amino acid sequence of SEQ ID NO: 2, a light chain CDR2having the amino acid sequence of SEQ ID NO: 3, a light chain CDR3having the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 6, a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 7, and a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 8; a light chain CDR1having the amino acid sequence of SEQ ID NO: 10, a light chain CDR2having the amino acid sequence of SEQ ID NO: 11, a light chain CDR3having the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 14, a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 15, and a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 16; a light chain CDR1having the amino acid sequence of SEQ ID NO: 18, a light chain CDR2having the amino acid sequence of SEQ ID NO: 19, a light chain CDR3having the amino acid sequence of SEQ ID NO: 20, a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 22, a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 23, and a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 24; or a light chain CDR1having the amino acid sequence of SEQ ID NO: 26, a light chain CDR2having the amino acid sequence of SEQ ID NO: 27, a light chain CDR3having the amino acid sequence of SEQ ID NO: 28, a heavy chain CDR1having the amino acid sequence of SEQ ID NO: 30, a heavy chain CDR2having the amino acid sequence of SEQ ID NO: 31, and a heavy chain CDR3having the amino acid sequence of SEQ ID NO: 32; further comprisingexposing the biological sample of the subject to a second antibody, orantigen-binding fragment thereof, comprising: a light chain CDR1 havingthe amino acid sequence of SEQ ID NO: 2, a light chain CDR2 having theamino acid sequence of SEQ ID NO: 3, a light chain CDR3 having the aminoacid sequence of SEQ ID NO: 4, a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 6, a heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 7, and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 8; a light chain CDR1 having the amino acidsequence of SEQ ID NO: 10, a light chain CDR2 having the amino acidsequence of SEQ ID NO: 11, a light chain CDR3 having the amino acidsequence of SEQ ID NO: 12, a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 14, a heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 15, and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 16; a light chain CDR1 having the amino acidsequence of SEQ ID NO: 18, a light chain CDR2 having the amino acidsequence of SEQ ID NO: 19, a light chain CDR3 having the amino acidsequence of SEQ ID NO: 20, a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 22, a heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 23, and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 24; or a light chain CDR1 having the amino acidsequence of SEQ ID NO: 26, a light chain CDR2 having the amino acidsequence of SEQ ID NO: 27, a light chain CDR3 having the amino acidsequence of SEQ ID NO: 28, a heavy chain CDR1 having the amino acidsequence of SEQ ID NO: 30, a heavy chain CDR2 having the amino acidsequence of SEQ ID NO: 31, and a heavy chain CDR3 having the amino acidsequence of SEQ ID NO: 32.